2016-2015

Manual 2015-2016 Editors: 1. Dr.Nehad Abdulmunem /Head of premarital lab, Shj 2. Dr.Eihab Abdelrahman/Premarital Clinic coordinator, Ajman PHC 3. Dr. Zainab Sabri/Premarital Clinic Coordinator, Shj PHC Reviewers: 1. Dr.SabriAl Sharqawi/Premarital Clinic Coordinator, Dubai PHC 2. Dr.Ohood Samhan/Premarital Clinic Coordinator, RAK PHC 3. Mr.Fouad Bin Abdulrahman, Shj PHC Supervisors: 1. Dr. Noura Al suwaidi /Former Head of Shj PHC and Premarital screening & counseling program 2. Dr. Fatima Al Sahi/Head of Premarital screening & counseling program

01 Table of Contents

Introduction 03

Procedure Index 04

05

10

Hepatitis B 11

Hepatitis C 12

HIV 14

Rubella 15

Syphilis 16

ABO/RH (D) Incompatibility 17

Abnormal findings 18

Diseases Guidelines 19

Appendices 45

References 73

02 Introduction • UAE Personal Status Act No. 28, Article 27, issued in June 2005, edicts that a compulsory premarital medical screening report has to be obtained by couples who are planning to marry in UAE.

personal status, communicate with MINISTRY OF JUCSTICE. In addition, approve the list of diseases in the screening program by the UAE CABINET for

develop policy and procedure, forms, and brochures.

this context, MOH is setting procedural standards for the premarital health screening required for issuing a Premarital Screening Report in the Emirates of Dubai, Sharjah, Ajman, Fujairah, Ras Alkhaima, umm al quwain.

primary health care and preventive health care and laboratories health facility in MOH. Scope:

1. To ensure couples receive effective health counseling and appropriate advice before marriage. 2. To diagnose previously undetected common medical, communicable and hereditary conditions in individual cases. 3. To protect the community and the unborn children from the financial, physical and psychological burden associated with the common communicable and hereditary conditions. 4. To provide counseling which might alleviates anxiety especially if there is a family history of certain genetic diseases or consanguinity. • Reaching these purposes requires a clinical and diagnostic service with high sensitivity, specificity and accuracy, completed within a limited turnaround time and with the highest integrity.

which must be met by any health care facility licensed to perform the Premarital Screening and Counseling Program.

Emirates. Staff members dealing with couples planning for marriage, as per their function, are primarily responsible for following these standards. Goal: To achieve a healthy society in UAE and prevent common diseases that can be transmitted through marriage to the partner and /or the offspring. 03 Procedure Index

Procedure No Procedure Name Revised Issue Date Number

MOH/PHC/PSC/01 e Overall process 26/04/2015 2.0

MOH/PHC/PSC/02 B-thalassemia & Significant 26/04/2015 2.0 haemoglobinopathies screening

MOH/PHC/PSC/03 Hepatitis B Screening 26/04/2015 2.0

MOH/PHC/PSC/04 Hepatitis C Screening 26/04/2015 2.0

MOH/PHC/PSC/05 HIV Screening 26/04/2015 2.0

Rubella immunity Screening MOH/PHC/PSC/06 26/04/2015 2.0 (for females only)

MOH/PHC/PSC/07 Syphilis Screening 26/04/2015 2.0

MOH/PHC/PSC/08 ABO/RH(D) incompatibility 26/04/2015 2.0

MOH/PHC/PSC/09 Other abnormal findings 26/04/2015 2.0

04 MOH / PHC/ PSC/ 01

No. Procedure Responsibility

1.1 - Premarital Screening Facilities treat all information of All Staff couples according code of ethics & professional conduct for civil services of the federal authority for government human resources.

1.1 - All Couples Planning to marry in UAE have to do the Administrator premarital screening and counseling. / clerk - In order to do the screening couple should attend any of the licensed premarital & counseling PHC centers together or separately.(see appendix 2, 1)

premarital screening service have to be ensured. - Documents required from Applicants applying for premarital screening are the followings: a. A verified copy of the original valid passport for Non-UAE Nationals and Emirates ID for the UAE and GCC nationals. b. 3 Colored standard passport size photos (4.5 cm X 3.5 cm) showing the full face. c. Payment fees are 100 AED for UAE nationals and GCC with Emirates ID and 250 AED for non- UAE & GCC nationals without Emirates ID, to be paid per partner according to MOH regulations. d. If applicant is below 18 years old, a guardian must be present. - Applicants identification data entry for the couples ( see Appendix 3) - Provide Education Booklet/CD for applicants.

05 and sign the premarital reports in case of normal results, and for that purpose a report collection delegation form should be signed by the Applicants. (see Appendix 4) - Recording applicant’s data in the registry record. - Receiving calls and answering queries about the service or results by Telephone. - Call the applicants to attend the center when the results/reports are ready if applicable.

1.3 - Measure the vital signs and record in Applicants file: Nurse weight, Height, BMI, BP, Pulse rate. - Deliver the Required vaccination according to physician request.

1.4 - Conduct the Pretest counseling which include the Physician followings: A /Advice couples on healthy life style and recomm- end female to take folic acid prior to pregnancy. B/ Counsel couples about what tests they are going to do and emphasize on the importance of that. - Each one of the couple should Sign a Premarital Applicant consent form. (see appendix 3) - Full history assessment and physical examination as mentioned in Appendix 3 - Request a laboratory test by signing the lab request form , documenting history of blood transfusion and adding any other comments .(see Appendix 5)

followings:

significant hemoglobinopathies(HbS,HbE,HbD,HbC, HbOArab, Hb lepore) B/ Screening for Complete blood picture (CBC)

06 C/ Screening to identify ABO & Rh(D) Blood type. D/ Screening to detect Infectious diseases (HIV,Hepatitis B ,Hepatitis C and Syphilis) E/ Screening to detect Rubella immunity for females. F/ Detect hepatitis B immunity status for assessment of hepatitis B positive partner and immunity after vaccination as well. - Request Vaccination of the following diseases as indicated (see Appendix 4) Rubella for eligible females. Hepatitis B for eligible partners.

1.5 - All samples collected at PHC center labs and sent to Lab Staff premarital screening lab. - All General Standards for Clinical laboratory Services in MOH should be followed. - A non-Fasting Blood sample collection is required from each one of the couple. - Sample collections will be carried out at the Facility Lab and specimens will be sent to the premarital screening Lab. - In case the sample will be sent later, facility lab will collect the sample according to( Appendix 8) - A second sample or confirmatory test could be requested by the premarital screening Lab if needed.

1.6 - Receive the Lab results and their Interpretation Physician authorized by lab specialist (usually within 3 working days and can be up to 10 working days in case of confirmatory test or after the second sample.)

1.7 - Results received should be documented in Applicants Physician file.(see appendix 4) - If one of the partners did the screening test at another heath center, then both a copy of his 1st visit record and the attested copy of the results should be available at one health center in order to issue the report.

07 - Positive cases record to be delivered to clerk/Admin for statistical purposes. If there is no abnormal results, proceed to issue and sign Premarital Screening & Counseling Reports. (See Apendix 6)

1.8 - If there is an abnormal result of a disease please follow Physician the next steps: 1. Discuss and counsel both of the couple individually then together after the agreement of the affected individual. 2. It is the responsibility of the attending physician to ensure that both of the couple is aware about the disease and its consequences before signing the report by couple. 3. If one of the couple refused to share the information about the disease or refused to sign the report then the report should not be issued and this must be recorded in the system or file.

guidelines for premarital risk assessment statements. 5. Refer to specialist for counseling if needed (According to MOH regulations). Proceed to issue & sign Premarital Screening & counseling report by couple after counseling, and record the risk assessment information in Arabic. (see Premarital Risk assessment statements in Appendix 9) 6. Counseling through calls or Delegation for report signing is not allowed. 7. Send the sealed report by facility messenger for judicial department for cases with risk of having disease for the partner or offspring.

1.9 Administrator premarital screening & counseling report copy and Lab / clerk results should be kept in each individual file at the premarital screening Clinic according the MOH policy.

the date of the test.

08 - In case one of the couple outside UAE and the marriage will be outside the country or by authorization of guardian ,issue the single report according the guidance written in the report. (see Appendix 7)

- If applicant want to Reissue another premarital report that will be according to the followings: A/ If the tests are still valid proceed to reissue another report without repeating the tests and sign by couple or their delegates. B/ If the tests are still valid and one of the partners have been changed proceed to do the premarital screening & counseling for the new one and after the results become ready issue the report and sign by couple or their delegates. Each report will hold the date of the sample collection for that partner. C/ If the tests are NOT valid for couple or one partner repeat the procedure from the beginning for the invalid one with new file and payment.

counseling and positive cases need to be recorded in monthly statistic form(Excel sheet) soft copy and to be send to main office of PHC at MOH.

09 MOH / PHC/ PSC/ 02

B-thalassaemia & Significant haemoglobinopathies screening

No. Process Responsibility

2.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

2.2 If the Lab results are Normal for B -thalassemia and Doctor Significant haemoglobinopathies, proceed to issue Premarital Screening & Counseling Reports if all other results are normal.

2.3 If there is abnormal result* for B -thalassemia or Doctor Significant haemoglobinopathies; Counsel the case separately & then together.

2.4 If Difficult case to counsel *, refer to genetic specialist Doctor counseling. Receive feedback report from the genetic specialist and counsel the cases separately and then together.

2.5 Issue & Sign Premarital Screening & Counseling Report by Doctor couple.

*Refer to the common and improtant variant hemoglobins guidelines (page 20 to 32)

10 MOH / PHC/ PSC/ 03

Hepatitis B Screening

No. Process Responsibility

3.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

3.2 If lab results are negative for hepatitis B proceed to issue Doctor Premarital Screening & Counseling Reports if all other results are normal.

3.3 If Lab results are positive do the followings: Doctor A/ Notify the preventive medicine department in the district. According to infection control policy. B/ Check the HBsAb titre for the HBsAg negative partner and in case not done collect second sample for it. C/ Counsel the case separately & if agree then counsel the case & partner together & provide the necessary health education. D/ Offer referral to specialist Physician for the HBsAg positive partner after counseling according to MOH/PHC rules and regulations (refer to Tips to counselor). E/ If the partner will not be informed, stop the process. F/ If they agree to continue and the titer ≥ 10 IU/L proceed to issue Premarital Screening & Counseling Reports, but if the titer < 10 IU/L proceed to the next step.

11 3.4 - Start vaccination immediately according to the Doctor following schedule: • 0,1,6 months (Preferred schedule) ,check antibody titer after at least 4 weeks after any dose (%50 after first%75- after second, protective antibody level according to CDC guidelines)

- If HBs Ab titer ≥ 10 IU/L after 4 weeks after any dose Doctor proceed to issue reports and recommend completing the vaccination series. - If HBs Ab titer < 10 IU/L, continue the series doses and check titer 4 weeks after last dose. 4. If AB titer <10mIU/m after 4 weeks from the last dose of vaccine series (0,1,6 months) recommend to restart the vaccine series.( 0,1,6 months) with checking the HBsAb titer after last dose of the 2nd series.

3.5 - If the couple requested to issue the premarital Doctor screening & counseling reports without waiting to check their immunity status at any stage, proceed to issue the reports with deciding the risk assessment statements according the Risk assessment statements for judicial department.

12 MOH / PHC/ PSC/ 04

HCV screening

No. Activity Responsibility

4.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

4.2 If the anti-HCV test result is Non-Reactive or Negative Doctor after the confirmatory test done at the premarital lab, proceed with issuing the Premarital Screening & Counseling Report (if all other results are normal)

4.3 If the Confirmatory test result for anti-HCV is positive do Doctor the followings: A/ Notify preventive medicine department in the district. B/ Counsel the case separately & if agree then counsel the case & partner together & provide the necessary health education. C/ Offer referral to specialist Physician for the anti-HCV positive partner after counseling according to MOH/PHC rules and regulations (refer to Tips to counselor). D/ Issue & Sign Premarital Screening & Counseling Report forms by couple.

4.4 If the partner will not be informed, stop the process. Doctor

13 MOH / PHC/ PSC/ 05

HIV Screening

No. Activity Responsibility

5.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

5.2 If the HIV screening test result is Non-Reactive or Negative Doctor after the confirmatory test done at the premarital lab, proceed with issuing the Premarital Screening & Counseling Report (if all other results are normal)

5.3 If the Confirmatory test result is positive do the followings: Doctor A/ Notify preventive medicine department in the district. B/ Counsel the case separately by reviewing Personal history from the patient again regarding occupation, sexual contact, any drug use and other risk factors. - If agree then counsel the case & partner together & provide the necessary health education. C/ If the partner will not be informed, stop the process.

5.4 Refer the case to Preventive medicine department in the Doctor district for further evaluation according to MOH regulations.

5.5 If the couple requested to issue the premarital screening & Doctor counseling reports sign forms by couple.

14 MOH / PHC/ PSC/ 06

Rubella Screening

No. Activity Responsibility

6.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

6.2 If IgG titer ≥ 15 IU/L (Immune) proceed to issue Doctor premarital screening & counseling report (if all other results are normal)

6.3 If IgG Titer < 15 IU/L ( Non Immune): Doctor • Counsel the case & partner together about vaccination. • Advice to avoid pregnancy for 1 month after the vaccination. • Get the consent signature of the couple in the consent form that they have been informed and given advice to avoid pregnancy for one month following vaccination( see Appendix 13) • Offer rubella or MMR vaccination (0.5ml) subcutaneous.

Proceed to issue & Sign Premarital Screening & 6.4 Doctor Counseling Report forms by couple.

15 MOH / PHC/ PSC/ 07

Syphilis Screening

No. Activity Responsibility

7.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

7.2 If the VDRL/RPR test result is Non-Reactive OR Doctor VDRL /RPR reactive and TPHA Negative proceed with issuing the Premarital Screening & Counseling Report (if all other results are normal).

7.3 If VDRL/RPR reactive and TPHA test is positive do the Doctor followings: A/ Notify preventive medicine department in the district. B/ Counsel the case separately & refer the case to dermatologist to do the clinical assessment and ensure the full treatment given. C/Receive a medical report from the dermatologist showing that the patient received the treatment. D/ If the partner will be informed, counsel the case & partner together & provide the necessary health education. E/ Issue & Sign Premarital Screening & Counseling Report forms by couple according the risk as follows: - If VDRL/RPR titer declined fourfold from baseline proceed to issue the report with no risk. - If titer not declined or treatment refused offer counseling and health education and issue the report according risk assessment.

16 MOH / PHC/ PSC/ 08

ABO/RH(D) incompatibility

No. Activity Responsibility

8.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

8.2 If the female is RH (D) negative and her partner is positive Doctor counsel them regarding the Anti-D according to MOH regulations.

8.3 Proceed to issue & Sign Premarital Screening & Doctor Counseling Report forms by couple.

17 MOH / PHC/ PSC/ 09

Other Abnormal Findings

No. Activity Responsibility

9.1 Receive Lab. results and Interpretation from premarital Doctor screening lab.

9.2 Counsel the case according to the other abnormal Doctor findings and refer the case to primary or secondary care accordingly.

9.3 Proceed to issue & Sign Premarital Screening & Doctor Counseling Report forms by couple

18 COMMON AND IMPORTANT VARIANT HEMOGLOBINS

19 Sickle Cell Hemoglobin (HB S) Sickle Cell Trait

GENOTYPE A/S Beta Chain Variant • Defined by Hb S heterozygosity. • Each red cell contains a mixture of approximately %60 Hb A and %40 Hb S • e amount of A in each cell is enough to prevent sickling under most physiological conditions

CLINICAL SYMPTOMS • Sickle cell trait is NOT associated with anemia. • Sickle cell trait offers some protection against malaria. • Occasional hematuria (blood in the urine) and hyposthenia (impaired renal concentrating ability) are associated with sickle trait. • Splenic infarction has been reported to occur at altitudes greater than 7,000 feet. • Some studies suggest that individuals with sickle cell trait are at a greater risk for sudden death under extreme conditions such as those that might occur during basic training in the military. ese conditions are: severe dehydration, malnutrition, physical overexertion and exhaustion. is risk though increased, is small.

PRECAUTIONS • Avoid hypoxic situations: Deep sea diving, flying in unpressurized aircraft, strenuous physical activity over a prolonged period of time.

COUNSELORS CHECK LIST Clinical indications: • Person is a healthy carrier. • Person is not sick. • Sickle cell trait is not a disease. • Sickle cell trait will not cause you to be anemic. • ere is a small amount of hemoglobin S, but not enough to change the shape of the red blood cell. • e red blood cells of a person with sickle cell trait remain round and flexible. Explain circumstances which might trigger cells to sickle: • High altitude in non-pressurized planes. • Other situations where one did not get sufficient oxygen for a long period of time. • Prolonged strenuous aerobic exercise such as in basic training for the military. Inheritance: • It is inherited, you can NOT catch it. • Sickle cell trait is not rare. • It is passed directly from parent to child. • It does not “skip” generations. If you have it that means one of your parents also has it. • It is not sex linked; meaning you may have gotten it from either your mother or your father. • Sickle cell trait will never change into sickle cell anemia. 20 CLINICALLY SIGNIFICANT SICKLING DISORDERS Sickle Cell Anemia (Hb SS)

GENOTYPE S/S Homozygous Beta chain variant • Hemoglobin S (%100-90) • Hemoglobin F may be slightly elevated

CLINICAL SYMPTOMS • Most severe form of sickle cell disease Clinical course variable • Severe anemia • Vaso-occlusion, pain episodes, organ damage • Aplastic episode, splenic sequestration, increased risk for infection • If HbF is greater than %10 there is a decreased risk of stroke See chart for other complications

PRECAUTIONS • Genetic counseling to clarify if any risk for child born with sickle cell disease. • Referral to High Risk OB Clinic for pregnancy. • Hypoxia, dehydration

SICKLE HEMOGLOBIN C/D/O Arab DISEASE (Hb SC/D/O Arab disease)

GENOTYPE S/C or D or O Arab Doubly heterozygous Beta chain variant • Co-inheritance of HbS and HbC/D/O Arab • Hemoglobin S and C/D/O Arab present in near equal amounts, • no HbA. • Hemoglobin F may be slightly elevated • Moderate to mild anemia Generally less severe than HbSS

CLINICAL SYMPTOMS • Inherit the gene mutation responsible for HbS from one parent, and the gene mutation for HbC/D/O Arab from the other parent. • Moderate to mild anemia • Generally less severe than HbSS • Fewer vaso-occlusive episodes • Retinal thrombosis and necrosis of femoral head more common • Spleen remains enlarged • Susceptibility to infection increased

PRECAUTIONS • Genetic counseling to clarify if any risk for child born with sickle cell disease. • Referral to High Risk OB Clinic for pregnancy. • Hypoxia, dehydration.

21 SICKLE HEMOGLOBIN BETA THALASSEMIA (Hb S/β-thalassemia)

GENOTYPE S/ β-thalassemia Doubly heterozygous Beta chain variant • Hemoglobin S predominates with small amount of hemoglobin F S/Beta alassemia0 (no beta chain production) • Hemoglobin S predominates with small amount of hemoglobin A in Sickle Beta+ alassemia • S/Beta alassemia+ (reduced beta chain production) having fewer complications than S/Beta alassemia0 or sickle cell anemia (SS).

CLINICAL SYMPTOMS • e clinical severity of S/β-thalassemia depends on the type of β-thalassemia mutation. If the patient is S/β0, then no normal globin chains are produced: one gene produces only the S version of the chain, and the other gene produces nothing. Like patients with HbSS, these patients have “only” HbS (plus HbA2 and HbF), and no HbA. e clinical and hematological findings are comparable to sickle cell anemia • If the patient is instead S/β+, then some functioning globin is produced, and therefore some Hb A is present. ese patients have a milder sickling syndrome than HbSS or S/ β0. • Differentiating S/β-0thalassemia from Hb SS requires not only HPLC but also additional information such as family or molecular studies.

PRECAUTIONS • Genetic counseling to clarify if any risk for child born with sickle cell disease or Beta thalassemia major • Referral to High Risk OB Clinic for pregnancy. • Hypoxia, dehydration

22 HEMOGLOBIN C Hemoglobin C Trait

GENOTYPE A/C Beta chain variant • Defined by Hb C heterozygosity. • Individuals have 25-40% C hemoglobin e rest is hemoglobin A. • Each red Cell contains a mixture of Hb A and Hb C. • Solubility : Negative (-) • Blood count normal • Moderate target cells on blood smear in the range 10-30% .

CLINICAL SYMPTOMS • No symptoms • Hemoglobin C trait is NOT associated with anemia.

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous CC Anemia or Sickle C Anemia

COUNSELORS CHECK LIST Clinical indications: • Person is a healthy carrier. • Person is not sick. • C trait is not a disease. • ere is a small amount of hemoglobin C, but not enough to change the shape of the red blood cell. • Sometimes persons have red blood cells that resemble a bulls-eye, we call those target cells. ey do not cause problems. Inheritance: • It is inherited, you can NOT catch it. • It is passed directly from parent to child. • It does not “skip” generations, if you have it that means one of your parents also has it. • It is not “sex-linked” meaning you may have gotten it from either your mother or your father.

23 CLINICALLY SIGNIFICANT HEMOGLOBIN C DISORDERS Hemoglobin C Anemia (Hb CC)

GENOTYPE C/C Homozygous Beta chain variant • Only C hemoglobin present. • For proper confirmation of this genotype. Both parents would have A/C Trait. • Solubility: Negative (-) • Marked increase in the number of target cells. • Hemoglobin C crystals, microspherocytes, osmotic fragility may be decreased.

CLINICAL SYMPTOMS • Anemia mild Jaundice intermittent Splenomegaly • Decreased red cell plasticity • Occasional episodes of joint and abdominal pain Aplastic events and gallstones may occur • No specific therapy is available or required for patients with Hemoglobin C Disease. Anemia may become more severe following infections, but the overall prognosis is considered to be excellent.

PRECAUTIONS • Genetic counseling to clarify risk for child born with hemoglobin C Disease, C/Beta alassemia or Sickle C Disease.

Hemoglobin C BETA THALASSEMIA (Hb C/β-thalassemia)

GENOTYPE C/ β-thalassemia Doubly heterozygous Beta chain variant • Individuals are double heterozygotes for hemoglobin C and Beta alassemia. C/Beta alassemia+ = %80-65 Hb C and %30-20 Hb A, and increased Hb F. C/Beta alassemia0 = No A with increased F and C. • Solubility: Negative (-) • Blood indices: decreased

CLINICAL SYMPTOMS • C/Beta alassemia+ = Mild anemia, low MCV value, and target cells. • C/Beta alassemia0 = Moderately severe anemia, splenomegaly, and possible bone changes. • May be misdiagnosed .a family study is essential for proper confirmation of this genotype

PRECAUTIONS • Genetic counseling to clarify risk for child born with possible Hemoglobin C Disease, C/Beta alassemia, or Sickle C Disease

24 Hemoglobin E Hemoglobin E Trait

GENOTYPE A/E Beta chain variant

• Individuals have 20-40% E hemoglobin the rest is hemoglobin A. • Each red cell contains a mixture of A and E. • May show elevated red cell count. • Occasional target cells on blood smear. • Slight hypochromia and mild microcytosis. • Red cell survival normal.

CLINICAL SYMPTOMS • No symptoms • Hemoglobin E trait is NOT associated with anemia. Clinically and hematologically normal.

PRECAUTIONS • Genetic counseling to clarify risk for child born with S/E Disease or Hemoglobin E/ alassemia.

COUNSELORS CHECK LIST Clinical indications: • Person is a healthy carrier of one gene for hemoglobin E • Person is not sick. • Hemoglobin E trait does not cause any medical problems • Hemoglobin E trait does not require any special medical care.

Inheritance: • Hemoglobin E is not rare • It is inherited, you can NOT catch it. • It is passed directly from parent to child. • If your child has hemoglobin E trait, this means at least one parent also has hemoglobin E.

25 CLINICALLY SIGNIFICANT Hemoglobin E DISORDERS Hemoglobin E Disease (Hb EE)

GENOTYPE E/ E Homozygous Beta chain variant

• Individuals have 90-98% E hemoglobin. ere may be a slight increase in Hb F. 130% Clinically well, with rare to moderate anemia,

CLINICAL SYMPTOMS • Clinically well, with rare to moderate anemia, microcytosis. Rare splenomegaly

PRECAUTIONS • Genetic counseling to clarify risk for child born with S/E Disease or Hemoglobin E/ alassemia.

Hemoglobin E β thalassemia (Hb E/β-thalassemia)

GENOTYPE E/ β-thalassemia Doubly heterozygous Beta chain variant • Individuals are double heterozygotes for hemoglobin E and Beta alassemia. • E/Beta alassemia+ = Individuals have 40% E hemoglobin 10-30% A, with a significant increase in Hb F (30-50% ) Slight hypochromia and mild microcytosis. Family studies necessary to confirm. • C/Beta alassemia0 =Individuals have E hemoglobin with a significant increase in Hb F (30-60% ) and no hemoglobin A

CLINICAL SYMPTOMS • E/Beta alassemia+ = Moderate anemia. Microcytosis, splenomegaly, jaundice • E/Beta alassemia0 = Severe disease with severe anemia Microcytosis, splenomegaly, jaundice, expansion of marrow space. Treatment is similar to homozygous beta thalassemia.

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous EE Disease, S/E Disease or Hemoglobin E/ alassemia .

26 HEMOGLOBIN D Hemoglobin D Trait

GENOTYPE A/D Beta chain variant • Defined by Hb D heterozygosity. • Individuals have 25-40% D hemoglobin e rest is hemoglobin A. • Each red Cell contains a mixture of Hb A and Hb D

CLINICAL SYMPTOMS • No symptoms• Hemoglobin C trait is NOT associated with anemia.

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous DD Disease, S/D Disease or Hemoglobin D/alassemia

Hemoglobin E β thalassemia (Hb E/β-thalassemia)

COUNSELORS CHECK LIST Clinical indications • Person is a healthy carrier of one gene for hemoglobin D • Person is not sick. • Hemoglobin D trait does not cause any medical problems • Hemoglobin D trait does not require any special medical care Inheritance: • It is inherited, you can NOT catch it. • It is passed directly from parent to child. • If your child has hemoglobin D trait, this means at least one parent also has hemoglobin D. • It does not “skip” generations, if you have it that means one of your parents also has it. • It is not “sex-linked” meaning you may have gotten it from either your mother or your father.

27 CLINICALLY SIGNIFICANT Hemoglobin D DISORDERS Hemoglobin D disease (Hb DD)

GENOTYPE D/D Homozygous Beta chain variant • Individuals have 90-98% D hemoglobin. • Clinically well, with rare to moderate anemia.

CLINICAL SYMPTOMS • Clinically well, with rare to moderate anemia. Rare splenomegaly

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous DD Disease, S/D Disease or Hemoglobin D/ alassemia • Family studies are necessary to confirm diagnosis.

Hemoglobin D BETA THALASSEMIA (Hb D/β+-thalassemia)

GENOTYPE D/ β+-thalassemia Doubly heterozygous Beta chain variant

• Individuals are double heterozygotes for D/Beta alassemia+ = 65-80% Hb D and %30-20 Hb A, and increased Hb F. • Slight hypochromia and mild microcytosis.

CLINICAL SYMPTOMS • Clinically well, with rare to moderate anemia, microcytosis. Rare splenomegaly

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous DD Disease, S/D Disease or alassemia • Family studies are necessary to confirm diagnosis

28 Hemoglobin D BETA THALASSEMIA (Hb D/β-0thalassemia ) GENOTYPE D/ β-0thalassemia Doubly heterozygous Beta chain variant • Individuals have D hemoglobin with a significant increase in Hb F and no hemoglobin A

CLINICAL SYMPTOMS • Clinically well, with rare to moderate anemia, microcytosis. Rare splenomegaly

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous DD Disease, S/D Disease or alassemia • Family studies are necessary to confirm diagnosis HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH) GENOTYPE A/D Beta chain variant • Individuals have 25-40% D hemoglobin • e rest is hemoglobin A. Red cell contains a mixture of A and D.

CLINICAL SYMPTOMS • No symptoms • Hemoglobin D trait is not a disease and does not cause any health problems.

PRECAUTIONS • Genetic counseling to clarify risk for child born with homozygous DD Disease, S/D Disease or Hemoglobin D/alassemia.

COUNSELORS CHECK LIST Clinical indications: • Person is a healthy carrier of one gene for hemoglobin D • Person is not sick. • Hemoglobin D trait does not cause any medical problems • Hemoglobin D trait does not require any special medical care Inheritance: • It is inherited, you can NOT catch it. • It is passed directly from parent to child. • If your child has hemoglobin D trait, this means at least one parent also has hemoglobin D. • It does not “skip” generations, if you have it that means one of your parents also has it. • It is not “sex-linked” meaning you may have gotten it from either your mother or your father. 29 VARIANT HEMOGLOBIN • Variant Hemoglobins is not a hemoglobin type (Variant “V/A”) • Hemoglobin type A with Variant “V” is not a type of hemoglobin, it is a collective term used to group those rare hemoglobin types that our screening test could not identify. • Most variants cause no medical problems or complications. • However, if the client is currently pregnant, or planning a family, you may want to have her partner tested. If the partner has sickle cell trait, they may be at risk for having a child with a serious hemoglobinopathy. • Mutations are constantly being defined. BETA THALASSEMIA MINOR (TRAIT) GENOTYPE A/BETA THAL Beta chain deletion • One gene for production of the usual amount of beta chains with the correct structure, and one gene for a decreased amount of beta chain production. • Individuals have %95-89 hemoglobin A with elevated hemoglobin A2 between %8.0-3.5. e normal range for A2 is between 1.7 and %3.4. Hemoglobin F may be present in the ranges of %5-1.5.

CLINICAL SYMPTOMS • Mild anemia and pallor • Slight splenomegaly in some carriers • Growth and development normal • Carrier may appear to have an iron deficiency anemia for which iron therapy is not effective.

PRECAUTIONS • Genetic counseling and families studies to clarify risk for child born with Sickle Beta thalassemia or Beta alassemia Major.

COUNSELORS CHECK LIST Clinical indications: • Person is a healthy carrier. • Person is not sick. • Beta thalassemia trait is not a disease. • is trait can cause you to have small, pale red blood cells. • is condition looks like an iron deficiency anemia. • No amount of iron supplementation will correct it. You should not take medicinal iron because your body will store it, causing other medical complications Inheritance: • It is inherited, you can NOT catch it. • It is passed directly from parent to child. • It does not “skip” generations, if you have it that means one of your parents also has it. • It is not “sex-linked” meaning you may have gotten it from either your mother or your father. Incidence: • Beta thalassemia trait is not rare • It has a worldwide distribution and is one of the most common hemoglobin variants identified. 30 BETA THALASSEMIA MAJOR COOLEYS ANEMIA

GENOTYPE BETA THAL Beta chain deletion • Blood smear: Hypochromic/microcytic RBC’s, fragmented RBC’s target cells, Howel-Jolly bodies, normoblasts, poikilocytosis, schitocytes, polychromasia. • Inherited defect in beta chain synthesis, resulting in practically no production of hemoglobin A with an increase in available alpha chains. • Individuals have 98-100% hemoglobin F with or without elevated hemoglobin A2.

CLINICAL SYMPTOMS • Jaundice, splenomegaly, bone malformations, pallor, weakness, failure to thrive, growth retardation, increased risk for infection, and shortened lifespan. • Severe anemia, client is transfusion and chelation therapy dependent. • Without treatment, death my result by age 4. • Generally less severe in the African American population than in the Mediterranean or Asian population.

PRECAUTIONS • e lifespan of individuals with Beta alassemia has been greatly increased due to chelation therapy, which rids the body of excess iron produced by chronic transfusions.

31 Notes on HBV virus: • Hepatitis B is a disease caused by the hepatitis B virus (HBV) it’s a DNA virus classified in the Hepadnaviridae family. • It is transmitted through percutaneous (i.e., puncture through the skin) or mucosal (i.e., direct contact with mucous membranes) exposure to infectious blood or body fluids. • HBV can cause chronic infection, resulting in cirrhosis of the liver, liver cancer, liver failure, and death. • Persons with chronic infection also serve as the main reservoir for continued HBV transmission • Hepatitis B vaccination is the most effective measure to prevent HBV infection and its consequences.

32 Pre-exposure Vaccination of Adults: • Primary vaccination consists of >3 intramuscular doses of hepatitis B vaccine.

produces a protective antibody response as follows: • Approximately %55–%30 of healthy adults aged <40 years after the first dose. • %75 after the second dose. • And >%90 after the third dose. • In older people vaccine protection is less when the age will be > 40 years old, in addition to age, other host factors (e.g., smoking, obesity, genetic factors, and immune suppression) contribute to decreased vaccine response.

Persons at risk for infection by sexual exposure:

• Sex partners of hepatitis B surface antigen (HBsAg) ¬positive persons. • Sexually active persons who are not in a long-term, mutually monogamous relationship (e.g., persons with more than one sex partner during the previous 6 months). • Persons seeking evaluation or treatment for a sexually transmitted disease. • Men who have sex with men.

Special recommendations and information about the vaccination:

• Testing should be performed 2–1 months after adminis¬tration of the last dose of the vaccine series using a method that allows determination of a protective concentration of anti-HBs (>10 mIU/mL). • Persons found to have anti-HBs concentrations of >10 mIU/mL after the primary vaccine series are considered to be immune. • Immunocompetent persons have long-term protection and do not need further periodic testing to assess antiHBs levels. • Immunocompromised persons might need annual testing to assess anti-HBs concentrations (see Booster Doses). • Persons found to have anti-HBs concentrations of <10 mIU/mL after the primary vaccine series should be revaccinated. Administration of 3 doses followed by anti-HBs testing 2–1 months after the third dose, usually is more practical than serologic testing after 1 or more doses of vaccine. • Persons who do not have a protective concentration of anti-HBs after revaccination should be tested for HBsAg.

33 o If the HBsAg test result is positive, the person should receive appropriate management, and any household, sex, or needle-sharing contacts should be identified and vaccinated (see Appendix C). o Persons who test negative for HBsAg should be considered susceptible to HBV infection and should be counseled about precautions to prevent HBV infection and the need to obtain HBIG postexposure prophylaxis for any known or likely parenteral exposure to HBsAg-positive blood.

• Booster doses are not recommended for persons with normal immune status who were vaccinated as infants, children, adolescents, or adults. Serologic testing is not recommended to assess antibody concentrations in any age group, except in certain circumstances.

Notes on HCV virus:

• Hepatitis C is a disease caused by the hepatitis C virus (HCV) it’s an Enveloped Positive-sense single-stranded RNA in the Flaviviridae family. • Chronic HCV infection develops in %85-%75 of HCV infected persons, about 15%25- of people will clear the virus from their bodies without treatment. And subsequently %70-%60 of people with chronic HCV develop liver disease and %2-%1 will get liver cancer and die. • It is most efficiently transmitted through large or repeated percutaneous exposure to infected blood (e.g., through injecting drugs) .Although much less frequent, occupational, perinatal, and sexual exposure can also result in transmission of HCV.

as a source of transmission to others.

34 Proceed to HCV testing as follows:

sample Indicate the rationale conversation for the test recommends that people your age be tested for hepatitis C. OR Hepatitis C is a virus that can cause gradual, progressive liver damage. People can have the infection for many years - even decades - without knowing it. Many people have no symptoms, so getting a blood test is the only way to know if Reassure patient about you have hepatitis C. the value of the HCV test exposed to the virus at any time in your life. obtain consent If it is all right with you, i would like to test you for hepatitis C today.

Interpretation of results of tests for HCV infection and further actions:

TEST OUTCOME INTERPRETATION FURTHER ACTIONS HCV antibody No HCV Sample can be reported as non reactive for HCV antibody. nonreactive antibody detected No further action required. If recent exposure in person tested is suspected test for HCV RNA HCV Presumptive A repeatedly reactive result is consistent with current antibody reactive HCV infection HCV infection, or past HCV infection that has resolved, or biologic false positivity for HCV antibody. Test for HCV RNA to identify current infection HCV Current HCV Provide person tested with appropriate counseling and link antibody reactive, infection person tested to care and treatment. HCV RNA detected HCV No current No further action required in most cases. If distinction antibody reactive, HCV infection between true positivity and biologic false positivity for HCV RNA not HCV antibody is desired, and if sample is repeatedly detected reactive in the initial test with another HCV antibody assay. In certain situations follow up with HCV RNA testing and appropriate counseling.

• If HCV RNA testing is not feasible and person tested is not immunocompromised, do follow-up testing for HCV antibody to demonstrate seroconversion. If the person tested is immunocompromised, consider testing for HCV RNA. • It is recommended before initiation antiviral therapy to retest for HCV RNA in a subsequent blood sample to confirm HCV RNA positivity. • If the person tested is suspected of having HCV exposure within the past 6 months, or has clinical evidence of HCV disease, or if there is concern regarding the handling or storage of the test specimen. 35 Providing a positive Hepatitis C result:

A positive hepatitis C test result should be communicated confidentially through personal contract by a clinician or trained health professional. When first hearing the test result, the patient may have a strong emotional reaction. e patient may want additional information immediately or may need some time to process what he/she has just heard. Take a cue from his or her reaction. It is important not to rush the patient after this initial disclosure.

sample Provide results i have your test result conversation If antibody test result Your hepatitis C test result were positive. You have hepatitis C. and HCV RNA test are both positive

If the patient is upset or scared about the test result, you may want to convey a positive message of hope about hepatitis. C.

sample Convey a positive • Many people with hepatitis C remain healthy conversation message throughout their lives. • ere are treatments available that can cure hepatitis C for many people. • ere is a lot you can do to keep yourself healthy. • You can find out if you have liver damage. • You can start doing things to take care of your liver and prevent more damage. • You can prevent transmitting the virus to others.

36 Counseling for daily living with Hepatitis C:

sample Talk about daily living Let

NB: CDC recommend all persons identified with HCV infection should receive a brief screening and interventions as clinically indicated to reduce or avoid alcohol consumption.

37 Notes on Rubella: INTRODUCTION

Rubella virus is classified as togavirus genus rubivirus , ( RNA Virus) Singal antigenic type that doesnot cross or react with other members of the togavirus Group. • Respiratory transmission virus • Incubation period 14 days (1213- days) • Viremia 57-days after exposure (transplacentalinfection of the fetus during viremia)

Clinical feature of rubella: 25 TO %50 Of victims are sub clinical .

• Mild fever • Maculopapular rash • Lymph adenopathy( Post auricular and suboccipital) • Malise

Objectives Of Rubella Screening and ImmunuzationProgramme: Miscarriage, still birth and fetal anomalies are the result from Rubella infection in early pregnancy. syndrome is infection of the fetus , which can affect all organ of the fetus . It can cause deafness ,cataract, heart defect, microcephaly , mental retardation, liver , bone and spleen damage.

Screening: It is done for all the female in the premarital programme. We check for immunoglobulin IgG titer.

Interpretation of titer • ≥15IU/ML it’s considered as immune. • <15 are none immune.

38 Counseling: • If immune, no need for vaccination and proceed to other session of premarital programme. • If none immune counsel the couple about vaccination and write consent not to be pregnant for at least 4 weeks after vaccination signed by couple. • Order rubella vaccine or MMR Vaccine as 0.5ml subcutaneous or Intra muscular. Caution: not to give vaccine for contraindication as allergic to vaccine , pregnancy, and immune compromised.

Notes on HIV virus: • HIV infection typically begins with a brief acute retroviral syndrome, transitions to a multi-year chronic illness that progressively depletes CD4 T- lymphocytes critical for maintenance of effective immune function, and ends with symptomatic, life-threatening immunodeficiency.

(AIDS), develops over months to years with an estimated median time of approximately 11 years. • In the absence of treatment, virtually all persons with AIDS will die from AIDS-related causes; however with antiretroviral therapy, persons provided early effective treatment can expect to live a near normal lifespan. • Early diagnosis of HIV infection and linkage to care are essential not only for the patients’ own health but also to reduce the risk for transmitting HIV to others. As of March 2012, U.S. guidelines recommend all persons with HIV infection diagnoses be offered effective antiretroviral therapy.

Common causes of HIV transmission: • HIV is mainly spread by having sex or sharing syringes and other injection equipment with someone who is infected with HIV. • Only certain fluids—blood, semen, pre-seminal fluid, rectal fluids, vaginal fluids,

must come in contact with a mucous membrane or damaged tissue or be directly injected into the bloodstream (from a needle or syringe) for transmission to possibly occur. Mucous membranes can be found inside the rectum, the vagina, the opening of the penis, and the mouth.

vaginal sex—but it is not zero.

ulcers, bleeding gums, genital sores, and the presence of other sexually transmitted diseases.

39 • Having sex with someone who has HIV. In general: o Anal sex is the highest-risk sexual behavior. o Vaginal sex is the second highest-risk sexual behavior. o Having multiple sex partners or having other sexually transmitted infections can increase the risk of infection through sex. • Sharing needles, syringes, rinse water, or other equipment (works) used to prepare injection drugs with someone who has HIV.

Less commonly, HIV may be spread by: • Being born to an infected mother. HIV can be passed from mother to child during pregnancy, birth, or breastfeeding.

mainly for health care workers. • Receiving blood transfusions, blood products, or organ/tissue transplants that are

blood supply and donated organs and tissues.

contamination occurs when infected blood from a caregiver’s mouth mixes with food while chewing, and is very rare. • Being bitten by a person with HIV. Each of the very small number of documented cases has involved severe trauma with extensive tissue damage and the presence

• Contact between broken skin, wounds, or mucous membranes and HIV-infected

rare. • Deep, open-mouth kissing if the person with HIV has sores or bleeding gums and blood is exchanged. HIV is not spread through saliva. Transmission through kissing alone is extremely rare.

Methods to reduce transmissions:

• Avoiding exposing partner to blood, semen, vaginal secretions, and other body fluids that are visibly contaminated with blood. • Avoiding contact with body fluids of HIV-infected persons after invasive oral or dental procedures. • Reducing the number of sex partners. • Communicating with partners to foster healthy sexuality (e.g., negotiating safer behaviors)

40 • Methods that HIV-discordant couples can use to reduce the risk of sexual HIV transmission, including the following; using latex and polyurethane male and female condoms: negotiating with partner to use; reminders to use; correct and consistent use. • Using only water-based spermicides and lubricants that do not contain nonoxynol9-. • Using dental dams or other physical barriers while having oral-vaginal or oral rectal sex. • Methods to prevent unintended pregnancy. • Conception options that reduce the risk of HIV transmission. • Interventions to reduce the risk of perinatal transmission. • Evidence that male circumcision may reduce a man’s risk of acquiring HIV from female partner with HIV. • Health benefits of abstaining from or reducing substance use, Risk of transmitting HIV when sharing drug-injection equipment.

Notes on syphilis: divided into stages based on clinical findings, helping to guide treatment and follow-up. Persons who have syphilis might seek treatment for signs or symptoms of primary syphilis infection (i.e., ulcers or chancre at the infection site), secondary syphilis (i.e., manifestations that include, but are not limited to, skin rash, mucocutaneous lesions, and lymphadenopathy), or tertiary syphilis (i.e., cardiac, gummatous lesions, tabes dorsalis, and general paresis). Latent infections (i.e., those lacking clinical manifestations) are detected by serologic testing. Latent syphilis acquired within the preceding year is referred to as early latent syphilis; all other cases of latent syphilis are late latent syphilis or syphilis of unknown duration. T. pallidum can infect the central nervous system and result in neurosyphilis, which can occur at any stage of syphilis. Early neurologic clinical manifestations (i.e., cranial nerve dysfunction, meningitis, stroke, acute altered mental status, and auditory or ophthalmic abnormalities) are usually present within the first few months or years of infection. Late neurologic manifestations (i.e., tabes dorsalis and general paresis) occur 30–10 years after infection.

41 Diagnostic Considerations

Darkfield examinations and tests to detect T. pallidum directly from lesion exudate or tissue are the definitive methods for diagnosing early syphilis (395). Although no T. pallidum detection tests are commercially available, some laboratories provide locally developed and validated PCR tests for the detection of T. pallidum DNA. A presumptive diagnosis of syphilis requires use of two tests: a nontreponemal test (i.e., Venereal Disease Research Laboratory [VDRL] or Rapid Plasma Reagin [RPR]) and a treponemal test (i.e., fluorescent treponemal antibody absorbed [FTA-ABS] tests, the T. pallidum passive particle agglutination [TP-PA] assay, various enzyme immunoassays [EIAs], chemiluminescence immunoassays, immunoblots, or rapid treponemal assays). Although many treponemal-based tests are commercially available, only a few are approved for use in the United States. Use of only one type of serologic test is insufficient for diagnosis and can result in false-negative results in persons tested during primary syphilis and false-positive results in persons without syphilis. False-positive nontreponemal test results can be associated with various medical conditions and factors unrelated to syphilis, including other infections (e.g., HIV), autoimmune conditions, immunizations, pregnancy, injection-drug use, and always receive a treponemal test to confirm the diagnosis of syphilis.

Nontreponemal test antibody titers might correlate with disease activity and are used to follow treatment response. Results should be reported quantitatively. A fourfold change in titer, equivalent to a change of two dilutions (e.g., from 1:16 to 1:4 or from 1:8 to 1:32), is considered necessary to demonstrate a clinically significant difference between two nontreponemal test results obtained using the same serologic test.

Sequential serologic tests in individual patients should be performed using the same

RPR are equally valid assays, but quantitative results from the two tests cannot be compared directly because RPR titers frequently are slightly higher than VDRL titers. Nontreponemal test titers usually decline after treatment and might become nonreactive with time; however, in some persons, nontreponemal antibodies can persist for a long period of time, a response referred to as the “serofast reaction.” Most patients who have reactive treponemal tests will have reactive tests for the remainder of their lives, regardless of treatment or disease activity. However, %25–%15 of patients treated during the primary stage revert to being serologically nonreactive after 3–2 years (397). Treponemal antibody titers do not predict treatment response and therefore should not be used for this purpose.

42 Some clinical laboratories are screening samples using treponemal tests, typically by algorithm for syphilis testing can identify persons previously treated for syphilis, those with untreated or incompletely treated syphilis, and persons with false-positive results that can occur with a low likelihood of infection. Persons with a positive treponemal screening test should have a standard nontreponemal test with titer performed reflexively by the laboratory to guide patient management decisions.

If the nontreponemal test is negative, the laboratory should perform a different treponemal test (preferably one based on different antigens than the original test) to confirm the results of the initial test. If a second treponemal test is positive, persons with a history of previous treatment will require no further management unless sexual history suggests likelihood of re-exposure. In this instance, a repeat nontreponemal test in 4–2 weeks is recommended to evaluate for early infection.

Unless history or results of a physical examination suggest a recent infection, previously untreated persons should be treated for late latent syphilis. If the second treponemal test is negative and the epidemiologic risk and clinical probability for syphilis are low, further evaluation or treatment is not indicated. Two studies demonstrate that high quantitative index values from treponemal EIA/CIA tests correlate with TPPA positivity; however, the range of optical density values varies among different treponemal immunoassays, and the clinical significance of these findings warrant further investigation (400,401).

For most persons with HIV infection, serologic tests are accurate and reliable for diagnosing syphilis and following a patient’s response to treatment. However, atypical nontreponemal serologic test results (i.e., unusually high, unusually low, or fluctuating titers) might occur regardless of HIV-infection status. When serologic tests do not correspond with clinical findings suggestive of early syphilis, presumptive treatment is recommended for persons with risk factors for syphilis, and use of other tests (e.g., biopsy and PCR) should be considered.

Further testing is warranted for persons with clinical signs of neurosyphilis (e.g., cranial nerve dysfunction, auditory or ophthalmic abnormalities, meningitis, stroke, acute or chronic altered mental status, and loss of vibration sense). Laboratory testing is helpful in supporting the diagnosis of neurosyphilis; however, no single test depends on a combination of cerebrospinal fluid (CSF) tests (CSF cell count or protein and a

43 reactive CSF-VDRL) in the presence of reactive serologic test results and neurologic signs and symptoms. CSF laboratory abnormalities are common in persons with early syphilis and are of unknown significance in the absence of neurologic signs or symptoms (402).

CSF-VDRL is highly specific but insensitive. In a person with neurologic signs or symptoms, a reactive CSF-VDRL (in the absence of blood contamination) is considered diagnostic of neurosyphilis. When CSF-VDRL is negative despite the presence of clinical signs of neurosyphilis, reactive serologic test results, and abnormal CSF cell count and/or protein, neurosyphilis should be considered. In this

CSF FTA-ABS test is less specific for neurosyphilis than the CSF-VDRL but is highly sensitive. Neurosyphilis is highly unlikely with a negative CSF FTA-ABS test, especially among persons with nonspecific neurologic signs and symptoms (403).

Among persons with HIV infection, CSF leukocyte count usually is elevated (>5 white blood cell count [WBC]/mm3). Using a higher cutoff (>20 WBC/ mm3) might improve the specificity of neurosyphilis diagnosis (404).

Treatment Penicillin G, administered parenterally, is the preferred drug for treating persons in aqueous crystalline), dosage, and length of treatment depend on the stage and clinical manifestations of the disease. Treatment for late latent syphilis and tertiary syphilis require a longer duration of therapy, because organisms theoretically might be dividing more slowly (the validity of this rationale has not been assessed). Longer treatment duration is required for persons with latent syphilis of unknown duration to ensure that those who did not acquire syphilis within the preceding year are adequately treated. Selection of the appropriate penicillin preparation is important, because T. pallidum can reside in sequestered sites (e.g., the CNS and aqueous humor) that are poorly accessed by some forms of penicillin. Combinations of benzathine penicillin, procaine penicillin, and oral penicillin preparations are not considered appropriate for the treatment of syphilis. Reports have indicated that practitioners have inadvertently prescribed combination benzathine-procaine penicillin (Bicillin C-R) instead of the standard benzathine penicillin product (Bicillin L-A) widely used in the United States. Practitioners, pharmacists, and purchasing agents should be aware of the similar names of these two products to avoid using the inappropriate combination therapy agent for treating syphilis (405). through clinical experience even before the value of randomized controlled clinical syphilis are based not only on clinical trials and observational studies, but many decades of clinical experience. 44 Appendix (1)

Premarital counseling Health Center Names Ministry of Health

Name of Name of Health Center Phone Number Fax Number Medical Area

Dubai AL ETIHAD 042692498 043449418

HOR AL ANZ 042683335 042651607

ALREFAH 043930777 043932777

AL MUHAISNAH 042886644 042886644 065220525 Sharjah ALRIQA 065244079 065249035 ALKALIDIYA 065286862 065283872

KOR FAKAN 092384664 092370170

AL MADAM 068821122 068821146

AL DHAID 068822693 068826113 7010231 Ajman AL MADINA 067422227 067441144 MUSHERIF 067030425 067400633 Umm Al Quwain AL KHAZAN 067660399 067660558 FALAJ AL MULLA 068824851 068824544

Ras Al Khaimah RAS AL KHIMAH 073421005 072336084

ALKEDRA 068838803 068838102

GULFAR 072233317 072233512

Fujairah MRISHID 092227099 092220864

45 043449418 042692498

042651607 042683335

043932777 043930777

042886644 042886644 065220525 065249035 065244079 065283872 065286862

092370170 092384664

068821146 068821122

068826113 068822693 7010231 067441144 067422227  067400633 067030425  067660558 067660399 068824544 068824851

072336084 073421005

068838102 068838803

072233512 072233317

092220864 092227099

46 Together For a Healthy Family Appendix (2)

Premarital Screening & Counseling Program

Region Name of the Clinic Contact Number Disease Prevention & Screening Center 6331300 02 Abu Dhabi Abu Dhabi Khalifa A Clinic 5561695 02 Bain Al JeserainClinic 5582900 02

Disease Prevention & Screening Center Al Ain 7635888 03 Eastern Al Muwaiji Clinic 7632200 03 Region Al Maqam Clinic 7684380 03 Oud Al Touba 7545110 03 Western Al Dhafra Family Medicine Centre in Madinat 8846227 02 List of Clinics Providing Premarital Screening Service Premarital of Clinics Providing List Region Zayed

Center/Clinic Telphone Number

Al Safa H.C 5021401-04

Al Mankhool H.C 5021201-04

Al Mamzar H.C. 5021801-04

Al Khawaneej H.C 5023001-04

Nad Al Hamar H.C 5023701-04

Al Mizher H.C 5022601-04

Al Towar H.C 5022101-04

Al Badda H.C 5081000-04

Al Qusais Clinic 5022001-04

Nad Al Shiba Clinic 5021601-04

Al Luaily 5022501-04

Abuhail Clinic 5021701-04

Umm Suqaim Clinic 5023101-04

47

Appendix (3)

Photograph PSC clinic Stamp Premarital Screening & Counseling Form( First visit)

ID/ Passport No Full Name

Nationality Sex Date of birth

Email. Res.No. 2 Mobile No, 1

Declaration of Consent I hereby give my consent to undertake premarital screening and counseling which include some genetic blood disease (sickle cell anemia andB- thalassemia, etc.) and some sexually transmitted diseases (hepatitis (B & C), AIDS and syphilis) in addition to the blood group , complete blood count, immunity to German measles for girls and any other tests that may be necessary .I . understand that the tests are limited and will not cover

fertility issue ;also, does not include the ability to have children free of diseases . I certify that the certificate will only be issued after discussing the results with me and 10 3 with my spouse and the court will look at the results. The working days as well as a second ١٠ to ٣ results take from blood sample may be needed to complete the tests...... : Applicant Signature:…………………………………… ...... : Admin Staff Signature:……………………………….... ) (All required documents& payments have been ensured and the ( applicant read the declaration before signing) Pretest Counseling Health Education & Life Style Yes No advise Given Socio-Demographic Data Education/ Occupation Consanguinity to the partner Risk Behavior Smoking No Yes Alcohol No Yes Substance Abuse No Yes (Specify) If Previously Married Did you have children No Yes Previous child with congenital No Yes (Specify) anomalies? Medical / Surgical H/O Sys Disease/ Current Medications No Yes (Specify)

H/O Surgeries No Yes (Specify)

H/O Blood Transfusion No Yes (Specify/ Last transfusion Date)

Relevant Physical Examination General Appearance Normal Abnormal Other Comments Family History for Genetic Diseases (If present)

Doctor Signature & Stamp: ------Date: ------48

PSC/F٠١

Appendix (4) Premarital Screening & Counseling Form (Second) Laboratory Investigations HPLC RPR / TPHA Positive Negative Hb. & indices HbsAg (Hep B) Positive Negative Blood Grouping & Rh HIV (Ag&Ab) Positive Negative Rubella (female) Immune Non- Immune Anti HCV (Ab). (Hep C) Positive Negative Anti HBs (if partner +ve) Immune Non- Immune Others (Specify) Counseling Assessment & Comments No Risk At Risk (specify) ------Management & Intervention Program Vaccination Recommended (Specify) ...... According to the following Schedule: ...... Given ( Date) Not Given( explain why)…………………………………………………… Name of No of Date vaccine doses

Contraceptive Methods Yes No Discussed ( If Indicated) Recommendation Treatment Yes No Revisit yes( date)………. No Referred (Specify) Notes:

*Rubella Vaccination Consent: : * I declare by signing this form that I was given full Advice to avoid getting pregnant during the first month After the date of getting the Rubella vaccination. ١month after vaccination the In case of pregnancy during the first Health care center will not take any Responsibility to the outcome of the pregnancy. : Client Signature: Partner signature: Vaccination Date:

Results were discussed in Results were discussed presence of other party separately (Positive Cases) Final Decision Certificate issued Date: Certificate not issued

Will hereby delegate the collection of my premarital screening report to: ------Name: ------Relationship: ------Or Name: ------Relationship: ------ID to be provided Signature of applicant: : Date:

Doctor Signature & Stamp: ------Date: ------49

PSC/F01

Appendix (5)

Premarital Laboratory Screening Form PSC Clinic Stamp Code No.: Requesting Doctor’s Name : Sign & Stamp Nationality: DOB : Passport No.: Sex : Address: Date : Time & Consanguinity: Work Tel : Date of Collection: Last Transfused: Mobile : Comment: Received Date.: Lab No.:

TEST RESULT RESULT FORM Hb. & Indices Blood Grouping / RH A / B / AB / O / Rh +ve -ve Normal Carrier Diseased Sickle Cell Normal Carrier Diseased Hb Variant: HbC Normal Carrier Diseased HbD Normal Carrier Diseased HbE Normal Carrier Diseased HIV 2+1 Negative Positive HBs Ag Negative Positive HBs Ab Immune Non Immune Anti-HCV Negative Positive RPR Non reactive Reactive VDRL Negative Positive Rubella IgG Immune Non Immune Comment: Reported, Date & Sign

Lab Stamp Reviewed By Analyst Signature

50 Appendix (6)

51 Appendix (7)

52 Appendix (8)

Appendix (8) - MANAGEMENT OF SPECIMENS FOR PREMARITAL SCREENING LABORATORY TESTING

INTRODUCTION Laboratory test results are dependent on the quality of the specimen submitted. If there is any doubt or question regarding the type of specimen that should be collected it is mandatory that the laboratory is called to clarify the order and specimen requirements. Purpose: To Provide steps to ensure that specimens for test are collected appropriately to maintain specimen identification integrity and biosafety standards. General: ■ Special care must be taken to protect samples from the effects of extreme temperatures and fluctuations. ■ Packaging of specimens for shipment must be designed to minimize breakage. ■ Rough handling of blood specimens may cause hemolysis and compromise test results. ■ Transfer of specimens to the laboratory should occur within as short a time period as possible.

Specimen Collection and Transport - General Considerations Safety Considerations 1. Follow universal precautions treating all specimens as potentially hazardous as required by MOH regulations. 2. All personnel handling specimens should use appropriate barrier protection including gloves. laboratory coat and gown. 3. Do not contaminate the external surface of the collection container and or its accompanying paperwork. 4. Minimize direct handling of specimens in transit from the collection site to the laboratory. Use plastic sealable bags with a separate pouch for laboratory slips or specific transport containers provided by the laboratory. Laboratory slips and other accompanying paper work should never come into contact with specimens.

General Guidelines for Specimen Collection 1. No special instruction such as fasting or special diets are required. Diurnal variation is not a major consideration.

53 2. Clearly label each specimen tube with the patient’s name. PMS code no. and date taken Unlabeled specimens cannot be processed for testing. 3. Lab PSC 001. Premarital Laboratory Screening Form.

The following information must be included. a. Client Information: Patient Name. Sex Date of Birth. Contact Phone Address. Code Number containing Submitting Clinic b. Client History: Consanguinity. last transfusion date. c. Physician Information: Ordering Physician Name. d. Specimen Information: Date Taken Time Taken (if applicable). Specimen Type (if applicable)

General Guidelines for Specimen Transport 1. Transport all specimens as specified above in item 4 of safety considerations. 2. Blood specimens for serological testing should be submitted in vacationers without anticoagulants. They should be refrigerated at 2 to 8 degrees Centigrade to prevent hemolysis and preserve specimen quality until they are delivered to the laboratory. 3. All specimens should be protected from temperature extremes during transport. Specimens should not be left in a hot. parked car and should bot be subjected to freezing temperatures unless a particular specimen requirement states that a specimen may be frozen.

Serology Tests:

Acceptable Specimens ■ 10 - 5ml of whole blood (red top tube) sufficient to yield a minimum of 0.5 ml of clear serum)

Specimen Transport/Storage ■ Specimens should be refrigerated until placed in the courier box for transport to the laboratory. ■ Specimens not delivered to the laboratory on the day of collection should be refrigerated at 2ºC to 8ºC ■ Extract the serum from the clot as soon as possible in order to avoid hemolysis. ■ The sample can be stored at 2ºC to 8ºC if the test is performed within 7 days or they may be deep-frozen at 20ºC.

54 ■ Sample that has been frozen and thawed more than 3 times should not be used. Unacceptable Specimens Reject and recollect when: ■ Specimens contaminated with bacteria. ■ Hemolysis are not satisfactory for testing. ■ Lipaemic serum. ■ Identification errors. Hematology Test Acceptable Specimens ■ 5 ml freshly collected blood in 2 EDTA vacutainers. Specimen Transport/Storage ■ Specimens run within four to eight hours of collections so if specimens not delivered to the laboratory on the day of collection CBC should be done. ■ Refrigerated (2º8-ºC) storage is recommended ■ Avoid exposure of the sample to ice, cold packs, extreme heat or extreme cold

Unacceptable Specimens Reject and recollect when: ■ Clotted ■ Old specimen ■ Hemolysis are not satisfactory for testing. ■ Insufficient sample ■ Identification errors ■ Tube contains more blood than the stated volume. Example: a capillary tube with blood above the 500 ul mark or a 2ml fill tube above the 2mL mark is over-filled. Polymerase Chain Reaction (PCR) for HCV RNA Acceptable Specimens ■ Specimens may be serum or plasma only. Blood should be collected in BD SST Serum Separator Tubes or sterile tubes using EDTA (lavender top) as the anticoagulant. ■ Required sample volume is 650 uL for the assay: 2.0 mL will permit repeat analyses as well as other testing.

55 Specimen Transport/Storage

■ Serum of plasma samples are collected aseptically to minimize hemolysis and bacterial. ■ Whole blood is stored at 225-.C for no longer than 6 hours. Tube manufacturer>s instructions are followed to separate serum or plasma from whole blood within 6 hours of collection. ■ Serum or plasma is transferred and stored in sterile 2 ml Nalgene cry vials or equivalent. ■ Plasma or serum may be transported at 28-.C or frozen at 20.C to 80.C. ■ Serum of plasma samples may be stored at 2.8.C for up to 3 days or frozen at 70.C colder in long-term storage. ■ Specimens should be stored in plastic vials and sealed tightly to prevent desiccation of the sample. ■ Serum and plasma samples may be frozen and thawed up to three times without a loss HCV RNA.

Unacceptable Specimens

Reject and recollect when: ■ Specimens are rejected if contaminated, hemolysis, or stored improperly. ■ Multiple freeze/thaw cycles of specimens. ■ Heat-inactivated specimens.

REFERENCES 1. CLSL Protections of Laboratory Workers from infections Disease Transmitted by Blood. Body Fluids and Tissue. Approved Guideline. CLSI Document M-29A Villanova. PA: CLSI. 1997. 2. Hepatitis Reference Laboratory. Centers for Disease Control and Prevention. National Center for HIV/AIDS. Viral Hepatitis. STD and TB Prevention. Division of Viral Hepatitis. Laboratory Branch SOP Title: Sample Handling Effective Date: 06200/. Approved by: Saleem Kamili. PhD and Jan Drobenive. MD. PhD

56 Appendix (9)

.1 •

.2 • 50% •

.3 • 100% •

.4 • 25% • 50% • 25% •

.5 • 50% • 50% •

.6 • 100% •

57 6 5 4

.1 •

.2

•

•

.3 • •

.4

•

•

58 Appendix (10)

Introduction

Disorders of hemoglobins, referred to as hemoglobinopathies,occupy a unique position in medical genetics. ey are the most common single-gene diseases inhumans, and they cause substantial morbidity. The World Health Organization has estimated that more than %5 of the world’s population are carriers of genes for clinically important disorders of hemoglobin.

THE HEMOGLOBINS

Structure and Function of Hemoglobin Hemoglobin is the oxygen carrier in red blood cells. e molecule contains four subunits: two α chains and two β chains. Each subunit is composed of a polypeptide chain, globin, and a prosthetic group, heme, which is an iron-containing pigment that combines with oxygen to give the molecule its oxygen transporting ability.

STRUCTURAL FORMULA FOR NORMAL HEMOGLOBIN

HEMOGLOBIN STRUCTURAL FORMULA

A Major Adult Hemoglobin 2 Alpha Chains + 2 Beta Chains

F Fetal Hemoglobin 2 Alpha Chains + 2 Gamma Chains

A2 Minor Adult Hemoglobin 2 Alpha Chains + 2 Delta Chains

Gene Dosage, Ontogeny, and Clinical Disease

e differences in gene dosage (four α-globin and two β-globin genes per diploid genome) and ontogeny of the α- and β-globins are important to an understanding of the pathogenesis of many hemoglobinopathies. Mutations in the β-globin gene are more likely to cause

59 disease than are α-chain mutations because a single β-globin gene mutation affects %50 of the β chains, whereas a single α-chain mutation affects only %25 of the α chains. On the other hand, β-globin mutations have no prenatal consequences because γ-globin is the major β-like globin before birth, and Hb F constitutes three quarters of the total hemoglobin at term. Because α chains are the only α-like components of all hemoglobins 6 weeks after conception α-globin mutations cause severe disease in both fetal and postnatal life.

THE HEMOGLOBINOPATHIES

e hereditary disorders of hemoglobin can be divided into three broad groups, depending on whether the mutation alters the globin protein, its synthesis, or globin developmental witching:

1. Structural variants, which alter the globin polypeptide without affecting its rate of synthesis; 2. alassemias, in which there is decreased synthesis (or, rarely, extreme instability) of one or more of the globin chains, resulting in an imbalance in the relative amounts of the α and β chains; and 3. Hereditary persistence of fetal hemoglobin, a group of clinically benign conditions that are of interest because they impair the perinatal switch from γ-globin to β-globin synthesis

Hemoglobin Structural Variants e hemoglobin structural variants can be separated into three classes, depending on the clinical phenotype 1. Variants that cause hemolytic anemia. e great majority of mutant hemoglobins that cause hemolytic anemia make the hemoglobin tetramer unstable. However, two of the best-known variants associated with hemolysis, sickle cell globin and HbC, are not unstable but cause the mutant globin proteins to assume unusual rigid structures. 2. Mutants with altered oxygen transport, due to increased or decreased oxygen affinity or to the formation of methemoglobin, a form of globin incapable of reversible oxygenation. 3. Variants due to mutations in the coding region that cause thalassemia because they reduce the abundance of the globin polypeptide. Most of these mutations impair the rate of synthesis of the mRNA or the protein. Some rare variants cause gross instability of the hemoglobin monomer, greater instability than that associated with the variants leading to hemolytic anemia.

60 alassemia: An Imbalance of Globin-Chain Synthesis

alassemias, collectively the most common human single-gene disorders, are a heterogeneous group of diseases of hemoglobin synthesis in which mutations reduce the synthesis or stability of either the α-globin or β-globin chain to cause α-thalassemia or β-thalassemia, respectively.

e resulting imbalance in the ratio of the α : β chains underlies the pathophysiological process. e chain that is produced at the normal rate is in relative excess; in the absence of a complementary chain with which to form a tetramer, the excess normal chains eventually

precipitate in the cell, damaging the membrane and leading to premature red blood cell destruction. e defect in hemoglobin synthesis also results in a hypochromic, microcytic anemia.

e α alassemias Genetic disorders of α-globin production affect the formationof both fetal and adult hemoglobinand therefore cause intrauterine as well as postnatal disease.

Clinical States Associated with α Thalassemia Genotypes

Number of α-Globin Gene α-Chain Clinical Condition Functional Genotype Production α Genes Normal 4 αα/αα 100% Silent carrier 3 αα/α − 75% α-alassemia trait (mild anemia, microcytosis) 2 α−/α− or αα/ − − 50% Hb H (β4) disease (moderately severe hemolytic 1 α−/− − 25% anemia) Hydrops fetalis or homozygous α-thalassemia (Hb 0% − −/− − 0 Bart’s: γ4)

61 CLASSIFICATION OF ALPHA SYNDROMES

GENE DELETION CLASSIFICATION TESTING METHOD Complications

Single gene deletion Remaining Silent Carrier Traces of Hb Barts at birth that three genes compensate almost Clinically and hematologically disappear. Diagnosed by completely. normal enumeration of the alpha genes by recombinant DNA technology. is is both technically difficult and expensive.

Deletion of two alpha genes Alpha alassemia Trait Traces of Hb Barts at birth that Mild anemia with small red disappear at 3-4 months of age cells. No evidence of iron deficiency A2 levels are normal

Deletion of three alpha genes Hemoglobin H Disease 5-20%Hemoglobin H Moderately severe microcytic hemolytic anemia resembling mild Cooley’s anemia. (See section on Hemoglobin H Disease)

Deletion of four alpha genes Fetal Hydrops Syndrome Hemoglobin Barts with small Severe hemolytic anemia amounts of Hemoglobin H and beginning in utero. Affected Portland. infants develop heart failure,

often stillborn between 34 and No hemoglobin A or F 40 weeks or dies within the first

hours of birth. e parents have a thalassemic Pregnant women carrying an blood picture with low MCV and infant with Fetal Hydrops MCH and normal hemoglobin Syndrome have a high rate of electrophoresis. severe toxemia of pregnancy and postpartum bleeding.

62 e β- alassemias

e β- alassemias share many features with α-thalassemia. Decreased β-globin production causes a hypochromic, microcytic anemia, and the imbalance in globin synthesis leads to precipitation of the excess αchains, which in turn leads to damage of the red cell membrane. In contrast to α-globin, however, the βchain is important only in the postnatal period.

Consequently, the onset of β-thalassemia is not apparent until a few months after birth, when β-globin normally replaces γ-globin as the major non-α chain, and only the synthesis of the major adult hemoglobin, Hb A, is reduced. e excess α chains are insoluble, so that they precipitate in red cells and their precursors leading to destruction of the red cells and ineffective erythropoiesis.

Because the δ gene is intact, Hb A2 production continues, and in fact, elevation of the Hb A2 level is unique to β-thalassemia heterozygotes. e level of Hb F is also increased, not because of a reactivation of the γ-globin gene expression that was switched off at birth, but because of selective survival and perhaps also increased production of the minor population of adult red blood cells that contain Hb F.

In contrast to α-thalassemia, the β-thalassemias are usually due to single–base pair substitutions rather than to deletions .In many regions of the world where β-thalassemia is common, there are so many different β-thalassemia mutations that persons carrying two β-thalassemia alleles are more likely to be genetic compounds than to be true homozygotes for one allele. Most individuals with two β-thalassemia alleles have thalassemia major, a condition characterized by severe anemia and the need for lifelong medical management.

Carriers of one β-thalassemia allele are clinically well and are said to have thalassemia minor. Such individuals have hypochromic, microcytic red blood cells and may have a slight anemia that can be misdiagnosed initially as iron deficiency. e diagnosis of thalassemiaminor can be supported by the level of Hb A2 (α2δ2)

63

CLASSIFICATION OF BETA SYNDROMES

TYPE TRAIT HOMOZYGOUS (Minor) (Major) Beta + alassemia Hemoglobin A2: increased Hemoglobin F Hemoglobin A: 10-50%

Beta 0 alassemia Hemoglobin A2: increased Hemoglobin F + HbA2 No hemoglobin A

Delta Beta alassemia Hemoglobin A2: normal or No hemoglobin A or A2 slightly reduced hemoglobin F: 100% hemoglobin F: 5-10%

Delta Beta Lepore 10-50%Lepore No hemoglobin A or A2 Hemoglobin F: 2-10% HbF and Lepore present

Normal hemoglobin concentrations are considered to fall within the following range: • Hemoglobin A = 95% • A= an elevated A2 would be greater than 3.5% (Note the exception of A2 levels in sickle cell trait, see Table 2) Also note with quantitation of Hb A2 by Micronchromatography, elevated levels of this hemoglobin (usually between 4.0% to 8.0%) generally indicate beta thalassemia trait F = <1% CBC (complete blood count) should be performed and reviewed. e mean erythrocyte corpuscular volume (MCV) is used as the first criterion of the screening protocol for thalassemia. Patients with microcytosis should be offered further testing to determine thalassemia carrier status.A low MCV is defined as:

• less than 80 for adults • less than 76 in children ages 4 to 7 years • less than 74 in children ages 18 months to 4 years • less than 70 in children ages 6 months to 18 months

• e A2 elevation associated with beta thalassemia trait may be suspected from careful inspection of the electrophoresis strip, but must be confirmed quantitatively. Electrophoresis on cellulose acetate medium at alkaline ph is a useful screening procedure for separating hemoglobin variants that are interacting with thalassemia and the hemoglobin of the thalassemia syndromes such as HbS. H, Barts, Constant Spring and Lepore. • Specimens that give results in the borderline or indeterminate range when quantitated by densitometry will usually give results that are clearly either normal or elevated when studied by chromatography.

Interferences

e hemoglobin variants C and E will co-elute on the micro-columns with Hb A 2.

Any sample with indicated A 2 levels greater than %10 should be checked by other assays such as electrophoresis to confirm the identity of abnormal hemoglobin (such as C or E). Iron deficiency can depress Hb A 2 levels in beta thalassemia carriers.

64 Variant Hemoglobins with alassemia Phenotypes

Hemoglobin E Hb E is a β-globin structural variant that causes thalassemia because the mutant β chain is synthesized at a reduced rate.

is allele is noteworthy for several reasons: its frequency, its allelic interaction with other β- globin mutants, and its effect on RNA splicing.

Although Hb E homozygotes are asymptomatic and only mildly anemic, individuals who are genetic compounds with an Hb E mutation and various β-thalassemia alleles have abnormal phenotypes that are largely determined by the severity of the other allele.

Complex alassemias and the Hereditary Persistence of Fetal Hemoglobin e large deletions that cause the complex thalassemias remove the β-globin gene plus one or more other genes from the β-globin cluster.

A group of large β-globin gene cluster deletions of medical significance are those that leave at least one of the γ genes intact . Patients carrying such mutations have one of two clinical manifestations, depending on the deletion: δβ°-thalassemia; or hereditary persistence of fetal hemoglobin (HPFH), a benign condition due to disruption of the perinatal switch from γ-globin to β-globin synthesis.

Homozygotes with either of these conditions are viable because the remaining γ gene or genes are still active after birth, instead of switching off as would normally occur. As a result, Hb F (α2γ2) synthesis continues postnatally at a high level, compensating for the absence of Hb A.

e clinically innocuous nature of HPFH is due to a substantial production of γ chains, producing a higher level of Hb F in heterozygotes (17%to 35% Hb F) than is generally seen in heterozygotes for δβº-thalassemia (5% to 18% Hb F). e deletions that cause δβº-thalassemia overlap with those that cause HPFH , and it is not clear why patients with HPFH have higher levels of γ gene expression.

65

Public Health Approaches to Preventing alassemia and Educational Genetic Counseling:

e initiation of carrier testing and prenatal diagnosis programs for thalassemia requires not only the education of the public and of physicians but also the establishment of skilled central laboratories and the consensus of the population to be screened.

Whereas population-wide programs to control thalassemia are inarguably less expensive than the cost of caring for a large population of affected individuals over their lifetime, the temptation for governments or physicians to pressure any population into accepting such programs must be avoided, and the cultural and religious views of each community must be respected.

All clients who have been identified with a hemoglobin variant should be provided educational genetic counseling. is process involves the clear communication of the medical, psychological, social, and genetic factors related to the condition being discussed.

GUIDELINES FOR COUNSELING

Communicate a functional understanding of the particular hemoglobinopathy in question and the genetic mechanism by which it is produced.

Correct any misconceptions that exist about the disease and its relationship to the carrier state.

Present to couples “at-risk” for having a child with disease a thorough discussion of alternative reproductive options.

Communicate effectively and clearly the facts of the situation to the

Counselee in a way that can be clearly understood. Any new term that is introduced should be defined. Charts and audio-visuals and other visuals should be utilized.

Encourage the counselee to ask questions, express feelings.

Invite family members or a potential spouse to participate in your program’s education, and screening process, or make referral.

Offer follow-up counseling session.

Document session in a letter sent to the family and physician. 66

e burden is on the counselor to communicate the information needed by the counselee

to make his or her own reproductive decisions in an open, non- judgmental environment.

PREREQUISITE FOR COUNSELING: AN ACCURATE DIAGNOSIS

Counseling should not take place until accurate screening results are obtained from a competent laboratory, utilizing state of the art procedures (High Performance Liquid Chromatography, (HPLC), Cellulose Acetate Electrophoresis, Isoelectric Focusing (IEF), A2 and F quantitation).cells, oxygen cannot get to all parts of the body. Organsthen become starved for oxygen and are unable to function properly. Two main divisions exist in the classification of thalassemia, the major and minor forms. alassemiamajor represents the homozygous or disease state whereno chains are being produced. For example, beta thalassemia major (Cooley’ s Anemia)indicates no betachains are being produced, while beta thalassemia minor representsthe heterozygousor trait form that indicatesdecreased production by one gene andnormal production by the other gene.

Indications for screening:

1- Premarital Pre-marriage screening for haemoglobinopathies is not usual in world but for some religious/ ethnic groups pre-marital screening for β thalassaemia heterozygosity may be more acceptable than pre-conceptual or antenatal screening. Significant maternal haemoglobinopathies:

2- Pre-conception testing Pre-conceptual testing is important because it can be difficult to complete antenatal screening and fetal diagnosis within the first 12 weeks of pregnancy if the couple is unaware of the risk. e individuals concerned must be informed of the result, whether or not an abnormality is found. If an abnormality is detected (Hb variant or possible/probable thalassaemia), partner testing should be offered, according to the testing algorithm (see below), if appropriate. Pre-conceptual testing should always be performed in women being investigated for infertility and in those having assisted conception. If a woman is found to have or be a carrier for a significant haemoglobinopathy, the partner or should be tested, if appropriate, and the women given counselling.

3- Antenatal antenatal screening recommended for clinically significant haemoglobinopathies. Whichever screening method is applied, the laboratory must ensure a provisional report is available within three working days from sample receipt. 67

4- Newborn screening All newborn babies should be screened for SCD. Such screening should also be extended to babies under the age of 1 year not tested before. e main objective of the newborn screening Programme is to improve outcomes in SCD through early treatment and care. e screening Programme will also detect certain other variant hemoglobin by the analytical methods currently used. Additionally, the finding of Hb F only or of a very low percentage of hemoglobin A (<%5·1) on the newborn screen will identify the majority of babies with β thalassemia major. Further testing of samples that show a significant abnormality is required.

Assessment of abnormal screening results Variant hemoglobin of clinical relevance in this context are hemoglobin S, C, D-Punjab, E, H, Lepore and O-Arab. Detection of a hemoglobin variant. If a significant Hb variant is identified it should be confirmed by a suitable alternative method (e.g. hemoglobin electrophoresis acid and or alkaline or capillary electrophoresis if the initial method was HPLC) Raised Hb A2 percentage. If no relevant variant hemoglobin is identified, Hb A2 percentage is assessed. Hb A2 level of ≥%4 in the presence of a MCH <27 pg indicates heterozygosity for β thalassaemia. If the Hb A2 is apparently >%10 on HPLC, a diagnosis of Hb Lepore should be considered while a Hb A2 level apparently >%15 may indicate Hb E trait. Other variant hemoglobin also have a retention time similar to that of Hb A2 on HPLC. Raised Hb F percentage. In the context of an MCH <27 pg, an isolated raised Hb F of >%5 identifies possible heterozygosity for δβ thalassemia. In the presence of a normal MCH, hereditary persistence of fetal hemoglobin (HPFH) should be considered. In addition, certain conditions that are asymptomatic or have a mild phenotype will be identified and need to be subsequently distinguished from clinically significant abnormalities. For example, hemoglobin D Iran or hemoglobin G needs to be distinguished Hb E confirmatory testing should be performed

Laboratory methods High-performance liquid chromatography (HPLC)

High-performance liquid chromatography can be used for the quantification of hemoglobin S, A2 and F and for the detection, provisional identification and quantification of many variant hemoglobin. HPLC usually provides accurate quantification of Hb A2 and is therefore suitable for the diagnosis of β thalassemia trait. Automated HPLC systems are being used increasingly as the initial diagnostic method in laboratories with a high workload. In comparison with hemoglobin electrophoresis, HPLC has the following advantages: 1 e analyzers are automated, therefore require less time and permit processing of large batches. 2 Very small sample volumes (5 μl) are sufficient for analysis. 3 Quantification of normal and separated variant hemoglobin is available on every sample. 4 Provisional identification of a larger proportion of variant hemoglobin can be made. 5 δ chain variants (recognition of which is important in the diagnosis of β thalassemia heterozygosity) are more easily detected. 68

High-performance liquid chromatography usually separates hemoglobin A, A2, F, S, C, D- Punjab and G-Philadelphia from each other. However, both Hb E and Hb Lepore often co-elute with A2 (as other hemoglobin co-elute with A, S and F) but may be recognized by alternative techniques. HPLC has the disadvantage that it also separates glycosylated and other derivative forms of hemoglobin, which can make interpretation more difficult. For example, derivatives of hemoglobin S co-elute with hemoglobin A2, rendering its quantification inaccurate. Careful examination of every chromatogram is essential. As with every method of hemoglobin analysis, controls should be run with every batch. Identification of variants is only provisional, and unrelated second-line methods should be used for confirmation. If HPLC is used as the screening technique, it is essential to check and maintain the positions of the windows, which are used as the first stage identification of any variants found. is is generally done by adjusting the column temperature or the flow rate so that the Hb A2 peak appears at a standard time. is is just as important as the calibration of the Hb A2 and Hb F levels and should be checked daily. Appropriate controls should be included Sickle solubility test e kits for sickle cell solubility tests that are predominantly used in our laboratory as a second independent method for S hemoglobin detect hemoglobin S down to a concentration of 20% (and sometimes below; in some cases as low as 8%). e methods are therefore capable of detecting all cases of sickle cell trait even when there is coexisting α thalassemia trait and or iron deficiency anemia. False positives have been described in patients with high plasma protein levels (Canning & Huntsman, 1970) and in anaemic patients when double the volume of blood is used in the test (Arras & Perry, 1972; Lilleyman et al, 1972). e latter problem can be avoided, however, by using a more concentrated sample of blood or washing the red cells. Sickle solubility testing should be employed whenever an unknown hemoglobin is encountered’ by HPLC In general,

Selection of laboratory methods Screening for abnormal variants. e analytical procedures employed must be capable of detecting all the common clinically significant hemoglobin variants, i.e. S, C, D-Punjab, E, O-Arab and Lepore, and must be suitable for screening for alassemias. e following techniques can be used in screening for hemoglobin variants: • High performance liquid chromatography (HPLC) • Capillary electrophoresis (CE) • Cellulose acetate electrophoresis (CAE) Abnormal results should be confirmed by a different technique that is appropriate for the likely variant. Another technique that can be used for confirmation, besides those listed above, is acid agar or acid agarose electrophoresisalthough this is not suitable as a screening technique. Sickle solubility testing can be used asconfirmation of an initial screen that suggests the presence of sickle hemoglobin.

69

Screening for thalassemia. Methods used are red cell indices in conjunction with measurement of Hb A2 levels. Routine measurement of blood indices includes measurements of MCH and mean cell volume (MCV); it is recommended that MCH (≤27) is used to screen for thalassemia as this parameter is more stable than MCV. ese measurements are usually reported for all routine blood counts. Hb A2 is quantified by HPLC or microcolumn chromatography. Selecting an HPLC system. When choosing an HPLC system for use in screening for hemoglobin variants and thalassemia, some general considerations needed: Our laboratory cut-off Hb A2 of 4 % or above has been set as the action point in the diagnosis of carriers of β thalassemia. A value of %0·5 for Hb F has been set for the investigation of a raised fetal hemoglobin. e chosen system must therefore be able to measure Hb A2 and Hb F with accuracy and precision at these cut-offs and detect the hemoglobin variants as specified by the premarital screening Programme. Peaks should be clearly separated for accurate quantification. Laboratories should understand how integration takes place and be aware that peaks measured on sloping baselines or on shoulders of adjacent peaks are likely to be less reliable High-performance liquid chromatography systems must be able to detect Hb A2 variant peaks, due to α or δ chain variants, and quantify them accurately. ese should be added into the total Hb A2 percentage. In a person with an MCH below the cut-off point (<27 pg), further investigation will be required if the total Hb A2 is above 3.5%.

Problems with the measurement and interpretation of Hb A2 with many HPLC systems, Hb A2 is overestimated in the presence of Hb S. However, this is not a problem because in sickle cell trait the percentage of Hb A is greater than Hb S and the reverse is true in Hb S/β thalassemia. e Hb A2 may be lowered by up to 0.5% in cases of severe iron deficiency anemia. Hb A2 values >4.0% with normal indices may indicate β thalassemia trait with or without co-existing α thalassemia. In this case: • Re-analyses FBC • Repeat Hb A2 • Consider B12/folate deficiency, drugs, liver disease/alcohol or HIV infection (Alperin et al, 1977; Galacteros et al, 1993; Howard et al, 2005; Wilkinson et al, 2007). Hb A2 values ≤4.0% with normal red cell indices and a normal Hb F level can usually be regarded as normal, although some mild β thalassemia alleles (mainly in subjects of Mediterranean origin) are associated with an A2 of ∙.

Interpretation of results in the presence of iron deficiency Severe iron deficiency anaemia (Hb <80 g/l) can reduce the Hb A2 level slightly (by up to 0.5%). In premarital screening there is no justification for delaying the investigation for haemoglobinopathies whilst treating iron deficiency presumptively, as this will delay the process of identifying at-risk carrier couples, consider the results in the partner and arrange DNA analysis if there is any risk.

70

Recommendation Abnormal laboratory screening results should be confirmed by a different technique that is appropriate for the likely abnormality. Laboratories performing l screening should utilize methods capable of detecting significant variants and be capable of measuring Hb A2and Hb Fat the cut-off points required by screening Programme. Quantification of Hb A2by CAE plus scanning densitometry is not recommended. A sickle cell solubility test is not recommended as a primary screening tool Assessment of iron status may be useful in the interpretation of laboratory tests but should not delay partner testing. Laboratories should be aware of the conditions that may not be detected when using the Screening Programmes and also of the effect of blood transfusion on the interpretation of results. Quality assurance and laboratory standards External Quality Assessment External Quality Assessment (EQA) provides a long-term, retrospective assessment of laboratory performance, allowing laboratories to demonstrate consensus with their peers and providing information on inter-method comparability. Participation in EQA is required for laboratory accreditation and is an essential part of clinical governance. (NHS Sickle Cell and alassemia Screening Programme 2009).

Quality assurance and laboratory standards External Quality Assessment External Quality Assessment (EQA) provides a long-term, retrospective assessment of laboratory performance, allowing laboratories to demonstrate consensus with their peers and providing information on inter-method comparability. Participation in EQA is required for laboratory accreditation and is an essential part of clinical governance. (NHS Sickle Cell and alassemia Screening Programme 2009).

Indications for DNA referral DNA analysis is performed only in a limited number of cases if the couples at risk having a borderline A%3.9-3.5) 2) Standardized reporting Testing for haemoglobinopathies is a complex area because of the number of haemoglobin disorders that are detectable. However, because of the diversity of haemoglobin variants and thalassaemia syndromes, there will always be some situations that require further tests on different specimens or family studies before a conclusive diagnosis can be achieved. General notes on reporting screening results • e sample date must be given (this can be extremely important if a person has had a recent blood transfusion). • If a blood transfusion has been received within 4 months, misleading data and conclusions may result. is possibility should normally be mentioned when haemoglobinopathy results are reported. • Analytical fact should be separated from interpretative opinion. e factual results should be given first and should be followed by a clear conclusion, which may include recommendations. If there is likely to be a delay in producing a final result. • Similarly, if information on ethnicity/family origin is used, it should be stated in the report. 71

• As it improves clarity, the conclusion should always be given both in full text and in standard abbreviation form in parentheses. For example: Sickle Cell Carrier (AS) or Sickle Cell Anemia (SS). e convention recommended is for the Hb initials to be reported in the order of greatest to least percentage. • If no further action is required it may be helpful to say further testing is not indicated.

Limitations of haemoglobinopathy diagnosis Sensitivity/specificity. No technique can identify all abnormalities but the combined sensitivity/specificity of the HPLC and IEF techniques for haemoglobins present at the time of screening is approximately 99%. e pattern of haemoglobin variants is not unique however and whilst some will be clarified by using the second technique, unequivocal identification can usually only be made by DNA analysis or mass spectrometry. For example, Hb D will only be of significance if found to be Hb D-Punjab, but for the purposes of the screening programmes, further testing is only required if Hb D occurs in possible combination with HbS in an individual Recent blood transfusion. Hemoglobin interpretation is misleading after a recent blood transfusion and necessitates repeat testing after 4 months if a pre-transfusion sample has not been analyzed DNA testing or family study if possible to avoid the need for a repeat specimen at 4 months post-transfusion, Laboratory staff undertaking testing should utilize information from their laboratory computer records where a possible recent blood transfusion may have occurred.

Linkage of neonatal and premarital screening programmes he original commitment in the White Paper was to establish ‘a linked premarital and newborn screening Programme for Haemoglobinopathy and SCD’ (NHS 2000). A key objective for the linkage is to minimize the adverse effects of screening – anxiety, misunderstanding, inaccurate information, unnecessary investigation and irrelevant follow-up – and this can only be achieved by good communication between the two screening services.

Method of linkage All units are required to have a local policy applying national guidance on the integration of premarital and newborn screening programmes. Different regions will develop different systems for linkage, which should include some form of ‘partner at risk’ alert notification. Local agreement is required between screening laboratories as to which premarital screening should be notified to the newborn screening laboratory through electronic links It is equally important that a method is established for communicating positive results back to the relevant premarital screening services and laboratories. Recommendation A local written policy for linkage is required, including some form of ‘partner at risk’ alert mechanism. Good communication may help to reduce unnecessary anxiety, possible misunderstandings and missed diagnoses and allow detection and investigation of possible errors. 72

References:

3. CDC: National center for HIV/AIDS ,Viral hepatitis,STD and TB prevention, Division of viral hepatitis 2015 A guide to comprehensive hepatitis C counseling & testing 2015 http://www.cdc.gov/hepatitis/Resources/Professionals/PDFs/ CounselingandTestingPC.pdf.

4. CDC: Basic information about HIV. CDC: Recommendations for HIV prevention with adults and adolescents with HIV in the United States, 2014 : Summary for clinical providers, December 2014 ,11 CDC: HIV Infection: Detection, Counseling, and Referral, 2015 STD treatment guidelines.

395) .5(http://www.cdc.gov/std/tg2015/references.htm395#), (http://www.cdc.gov/std/tg2015/references.htm396(395# (http://www.cdc.gov/std/tg2015/references.htm395#)). http://www.cdc.gov/std/syphilis.

6. http:www.cdc.gov/hepatitis b

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