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PARTICIPANT STATISTICS CELL IDENTIFICATION THIRD QUADRIMESTER 2020
Q3-2020 Specimen Integrity and Interference from Cryoglobulin
Continuous advances in instrument technology, computer science, and automation both simplify laboratory diagnostics and reduce analytical error rates. Widespread use of automated hematology analyzers has enhanced the accuracy of test results and significantly reduced the turnaround time of the CBC. Significant portions of both time and money are dedicated to performing and documenting all aspects of laboratory testing quality assurance. However, occasionally inaccurate results are reported even when the testing equipment is functioning perfectly.
Laboratory testing is divided into three phases: preanalytical, analytical and postanalytical. In comparison to the analytical and postanalytical phases, the preanalytical phase is much more vulnerable to uncertainties and accidents. The preanalytical phase includes the time from the order of the test by the clinician until the sample is ready for analysis. A fundamental laboratory axiom is “ The patient test results are only as good as the specimen that is tested “.
Nearly 70% of laboratory errors are the result of pre-analytical issues. They are not actually laboratory errors in the sense that a laboratorian made a mistake or an instrument generated an incorrect result, but rather they are really specimen integrity issues.
Preanalytical errors include two types of variables, the first are sample related variables, such as hemolysis, sample collection technique, transport, and storage. The second are patient related variables. These include recent exercise, stress, age, sex, positional effects, menstruation and unique patient specific sample characteristics.
Manufacturer’s instrument manuals and in-house developed laboratory procedures detail the kinds of specimen characteristics that are known to interfere with accurate laboratory testing. In the case of hematology analyzers, cryoglobulins, lipids, insufficiently lysed RBCs, NRBCs and platelet clumping are common situations affecting WBC counts. In addition, lipemia, agglutinination, cryoglobulins and extremely elevated WBC counts can interfere with the accuracy of one or more CBC parameters. The flagging capabilities, WBC scattergrams and RBC and platelet histograms generated by the more sophisticated hematology analyzers will alert the operator to many of these interferences. Less complex analyzers, especially those without a WBC differential scattergram, do not have the same sensitivity for detecting spurious results.
These advancements in hematology analyzers have dramatically reduced blood smear review rates. Ironically, as more data on spurious results generated by these instruments is becoming available, the importance of the blood smear examination is again being recognized. Blood smear review has historically been used both as a quality assurance check and as a means of providing additional clinically relevant information.
In this case study, a hematology analyzer generated WBC error flag. In this laboratory (as is the case in pretty much all laboratories), error flags require that the sample results be reviewed/investigated using alternate methods. Review of the blood smear was the alternate method selected as most likely to provide insight as to the cause of the flagging. The visual inspection of the blood smear showed the presence of strands of light blue-gray “strand-like” structures, morphology consistent for the presence of cryoglobulins.
The sample was warmed and retested. Another blood smear was made from the warmed blood. The rewarmed sample did not generate any error flag nor did the blood smear exhibit the suspicious “strand-like” structures seen on the original blood smear. Upon further investigation, it was determined that the sample had been refrigerated overnight and tested the following morning. The conclusion was that a pre-analytical sample characteristic had resulted in the interference.
Precipitation of blood proteins is referred to as cryoprecipitation. Two types of cryoprecipitates are recognized. When proteins precipitate from both serum and plasma they are cryoglobulin, but when proteins precipitate from plasma but not serum, they are cryofibrinogen.
Cryoglobulins are circulating immunoglobulins that precipitate in cold temperature between 4°C and 37°C and dissolve when heated back to 37°C. They may be monoclonal, polyclonal or mixed. Since otherwise healthy individuals can develop transient asymptomatic cryoglobulinemia following certain infections, individuals found to have circulating cryoglobulins, but no signs or symptoms of cryoglobulinemic diseases, should be evaluated for the possibility that their cryoglobulinemia is a transient response to a recent or resolving infection. Patients with a history of recent infection that also have a spontaneous and full resolution of their cryoglobulinemia need no further treatment.
However, cryoglobulinemia is associated with pre-malignant, malignant, infectious, and autoimmune diseases, meaning that individuals without a history of infection and not showing resolution of their cryoglobulinemia, need to be further evaluated.
Cryoglobulinemic disease is classically grouped into three types. Type I, which encompasses about 10-15% of diagnosed cases of cryoglobulinemic disease, exhibit monoclonal cryoglobulins and contain only one subclass of immunoglobulin, either IgM, IgG, IgA or rarely Bence Jones protein. Type I is associated with multiple myeloma, Waldenstrom’s macroglobulemia and other lymphoproliferative disorders with monoclonal proteins.
Signs and symptoms due to the cryoglobulins of Type I disease reflect the hyperviscosity and deposition of cryoglobulins within the blood vessels which reduce or stop blood perfusion to tissues. The interruption of blood flow to neurological tissues can cause symptoms of confusion, headache, hearing loss, and peripheral neuropathy. Interruption of blood flow to other tissues in Type I disease can cause cutaneous manifestations of purpura, blue discoloration of the arms or legs (acrocyanosis), necrosis and ulcers; spontaneous nose bleeds, joint pain, and cardiovascular disturbances such as shortness of breath, hypoxia, and congestive heart failure.
The most common diseases associated with Type II cryoglobulinemic disease are chronic active hepatitis C and HIV. Type II cases exhibit a mixture of monoclonal IgM plus polyclonal IgG or, rarely, IgA. This category represents 50-60% of diagnosed cases of cryoglobulinemic disease. The remaining 25-30% of these cases are Type III. This group exhibits polyclonal IgM plus polyclonal IgG or IgA. Type III is associated with autoimmune disease such as Sjogren syndrome and, less commonly, SLE, RA and infectious diseases particularly HCV infections.
Types II and III cryoglobulinemic disease may also present with symptoms and signs of blood hyperviscosity syndrome and deposition of cryoglobulins within blood vessels. In addition, approximately 30% these patients may develop cryoglobulinemic vasculitis, characterize by a set of symptoms, known as “Meltzer's triad", that includes palpable purpura, joint pain, and generalized weakness. These mixed Type II/III cases sometimes will present with any of the following symptoms: painful peripheral neuropathy (often manifesting as mononeuritis multiplex in 19-44% of cases), kidney disease (primarily membranoproliferative glomerulonephritis (30%), joint pain (28%), and, less commonly, dry eye syndrome and Raynaud’s phenomenon.
17Q3 Immunohematology Page 1 of 3 Cryofibrinogen is an abnormal complex of proteins that precipitate out of plasma as it is cooled. These cold, insoluble protein complexes can be composed of fibrin, fibrinogen, fibrin split products, along with other plasma proteins. Cryofibrinogen can occur spontaneously (ie, essential cryofibrinogenemia) or in association with other inflammatory conditions. Secondary cryofibrinogenemia has been reported in patients with malignancy, diabetes mellitus, collagen vascular disease, and active infection.
Cryofibrinogenemia can produce symptoms due to cutaneous ischemia including purpura, ecchymosis, ulcerations, or ischemic necrosis. These symptoms are typically observed in areas of the body that tend to be at lower temperature including the hands, feet, ears, nose, and buttocks. Although most individuals with cryofibrinogenemia are asymptomatic, there are cases of morbidity that are the result of occlusion of the small to medium arteries by insoluble protein complexes.
Patients exhibiting only mild symptoms of either cryoglobulinemia or cryofibrinogenemia, generally are advised to avoid exposure to the cold, especially fingers and toes. Treatments with a corticosteroid plus low-dose aspirin followed by maintenance therapy with an anabolic steroid where necessary are recommended for moderately severe cases. Very severe cases generally require an immunosuppressive drug regimen and, if extreme or life threatening, can require treatment with plasmaphoresis.
For patients with a specific underlying pre-malignant, malignant, infectious, or autoimmune disease, the patient will also receive treatment with the methods prescribed for the specific disorder.
Cell Identification
Specimen 11 Specimen 12 Specimen 13 Specimen 14 Specimen 15 Result No. Flag Result No. Flag Result No. Flag Result No. Flag Result No. Flag Monocyte, any stage 70 Platelet, giant 149 Fibrin, Fibrin Strands 149 Polychromatophilic RBC 112 Myelocyte 93 Immature Neutrophil 25 Hairy Cell 6 *** Stain artifact 10 *** Howell-Jolly Bodies 40 Immature WBC, would refer 18 Segmented Neutrophil (PMN, poly) 27 Abnormal Platelet, would refer 3 Abnormal, would refer 5 *** Heinz Bodies (supravital stain) 3 *** Promyelocyte 14 Metamyelocyte 17 Platelet, normal 3 *** Parasite 1 *** Erythrocyte, normal RBC 2 *** Lymphocyte, reactive 11 *** Monocyte, normal/any stage 12 Lymphocyte, normal 2 *** Yeast 1 *** Pappenheimer Bodies 2 *** Abnormal, would refer 7 PMN with Pelger-Huet Nucleus 4 Abnormal, would refer 2 Abnormal, would refer 1 Monocyte, any stage 6 *** Abnormal, would refer 3 Basophil, any stage 1 *** Immature RBC, would refer 1 *** Lymphocyte; atypical, Downey, variant 4 *** Immature WBC, would refer 2 Macrocytic 1 *** Abnormal Lymphocyte, would refer 3 *** Abnormal Granulocyte, would refer 2 Erythrocyte, normal RBC 1 *** Abnormal Granulocyte, would refer 3 Abnormal Lymphocyte, would refer 1 Reticulocyte (supravital stain) 1 *** Lymphocyte, normal 3 *** Myelocyte 1 Basophilic Stippling 1 *** Monocyte, normal/any stage 1 *** PMN with Toxic Granulation/Vacuolization 1 Abnormal Platelet, would refer 1 *** PMN with Degenerated Nucleus (pyknotic PMN) 1 Myeloblast 1 *** Lymphocyte, abnormal/atypical 1 ***
Total Population 166 Total Population 166 Total Population 166 Total Population 165 Total Population 166 Intended result: Monocyte any stage Intended result: Platelet, giant Intended result: Fibrin, Fibrin Strands Intended result: Polychromatophilic RBC Intended result: Myelocyte Due to a lack of participant consensus, Specimen 11 was not evaluaed.
Correct responses are defined as those reflecting agreement among 80% or more of all participants or referees. Unacceptable responses are indicated by "*****" on the Flagging line of each specimen.
17Q3 Immunohematology Page 2 of 3 Cell Identification - Educational Challenge
Specimen 11 No. Specimen 12 PMN with Toxic Granulation/Vacuolization 60 Eosinophil, any stage 112 PMN with bacterial inclusion 17 PMN with Toxic Granulation/Vacuolization 3 Segmented Neutrophil (PMN, poly) 19 Abnormal Granulocyte, would refer 2 Basophil, any stage 11 PMN with bacterial inclusion 2 Abnormal Granulocyte, would refer 5 Lymphocyte, normal 1 Eosinophil, any stage 4 Abnormal, would refer 1 PMN with Pelger-Huet Nucleus 2 Hypersegmentated Neutrophil 1 Eosinophil, any stage 1 PMN with Pelger-Huet Nucleus 1 Abnormal, would refer 1 Bacteria 1 PMN with Degenerated Nucleus 1 Total Population: 122 Total Population: 123 Intended result: PMN with Toxic Intended result: Eosinophil, any
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Cell ID Educational Case History 3Q 2020 History:
A 3-year-old boy is brought to the Emergency Department due to a 3-day history of fevers up to 104oF. His mother states he has a history of recurrent bacterial respiratory infections, most recently requiring hospitalization. In addition, he has been avoiding brightly lit areas, including outside in his yard. Currently, the child is refusing to eat after having vomited his last meal. On physical examination, the patient is thin, febrile, and hypopigmented patches are present over his face and limbs. There is mild pallor of the conjunctivae and nystagmus is evident. Cervical lymphadenopathy and mild hepatomegaly are noted. In addition to other laboratory tests, a complete blood count is performed. CBC results are as follows: WBC 9.9 x 103/µL, Hgb 8.2 g/dL, Hct 27.4%, Plts 88,000/µL.
Pending writeup
17Q3 Immunohematology Page 3 of 3