Renal Function Markers

Proper renal function is important in normal homeostasis as they excrete waste products and remove excess fluid through steps of excretion and reabsorption. The kidneys regulate the bodies electrolytes as well as produce hormones such as EPO that stimulates the bone marrow to produce erythrocytes. The kidneys produce a second hormone called renin from the juxtaglomerular cells located in the renal arteries. When renin is secreted it acts on angiotensinogen and converts it to angiotensin I. Angiotensin I is then converted to angiotensin II by angiotensin converting enzyme. Angiotensin II acts on blood vessels and causes vasoconstriction that raises blood pressure.

The kidneys play such an important role in normal physiology that its imperative that they are kept functioning properly. It is common to have screening tests done annually to evaluate renal function. It is not uncommon for a physician to order a renal function test to role out chronic kidney disease (CKD). There are few tests that are important and can paint a picture as to how well the kidneys are functioning. Blood urea nitrogen (BUN) provides a rough measurement of the GFR. Urea is formed in the liver as an end product to protein metabolism. It is a breakdown product from use of amino acids. In impaired renal function, the kidneys will inadequately excrete urea, which elevates blood BUN levels. Serum creatinine is another important indicator of renal health because it is solely excreted by the kidneys. Creatinine is a waste product created by muscle metabolism. Creatinine is synthesized via creatine, phosphocreatine, and adenosine triphosphate (ATP). Creatine is synthesized in the liver and is transported through blood to the other major organs where through phosphorylation is converted to phosphocreatine. Creatine becomes phosphocreatine through a catalytic reaction by creatine kinase. The by-product produced by that reaction is creatinine. Little to no tubular reabsorption of creatinine occurs so if there are elevated levels detected in the blood, it is an indicator of renal impairment. The creatinine levels in the blood and urine can be used to calculate the creatinine clearance which correlates to the GFR.

It is important to note that a creatinine concentration in urine may also be tested during a drug of abuse screen. Normal creatinine levels indicate a test sample is undiluted, therefore if there are decreased levels of creatinine it indicates a manipulated test and the test must be repeated.

The GFR describes the flow rate of filtered fluid through the glomerular capillaries into the Bowman’s capsule per unit time. Its important to note that a normal GFR level decreases with age so that must be taken into account when screening patients with suspected CKD, for example the reference range for GFR in adults age 20-29 is 116, in adults 60-69, the GFR should be around 85. A physician can also properly stage CKD based on ones GFR. A progressively decreasing GFR indicates disease progression and more aggressive treatment needs to be considered. GFR is measured typically using a patients creatinine level in accordance with there age, sex, and body size. There are multiple equations that can be used that have all been validated, but are slightly different. Certain physicians or hospitals may have standardized ways of calculating the GFR. There is the Bedside Schwartz equation which should be used for patients 18 years of age and younger. The Modification of Diet and Renal Disease (MDRD study equation and the Chronic Kidney Disease Epidemiology Collabortion (CKD-EPI) equation are the most commonly used for adults 18 and older.

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Physicians may order microalbumin testing to screen individuals who are at high risk for developing CKD, especially diabetics. A urine microalbumin test detects minute levels of albumin in the urine. Albumin is one of the first proteins that be detected in the urine when renal function becomes impaired. Albumin is part of the globular protein family whose main function is to regulate the colloid osmotic pressure. Albumin also serves as a protein carrier for hydrophobic molecules such as lipid-soluble hormones, unconjugated bilirubin, free fatty acids, as well as some types of particular drugs like warfarin and phenytoin.

The kidneys are arguably one of the most important organs in homeostasis and its important that they are functioning properly. There are number of tests that can be performed to test renal function with each one giving a little piece of the puzzle. Physicians can use these tests to rule out CKD, or stage a patients disease progression.

-Caleb

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Case Study Mini-Series; Diagnostic Process and Treatment

Diagnostic workup of a suspected patient with APL should include a case history and physical examination with focus on bleeding tendencies, recurrent infections and anemic symptoms such as fatigue or pallor. A complete blood count with a differential should be performed. During a peripheral blood smear the technologist should be looking for abnormal promyelocytes with abundant azurophilic granulation and multiple auer rods. A bone marrow aspirate with cytology, cytochemistry, immunophenotyping, FISH, RT-PCR, and cytogenetics should be included. Diagnostic coagulation tests such as PT, aPTT, fibrinogen, and a D-dimer should be performed. During the immunophenotyping the characteristic phenotype of APL is CD33, CD13, CD45, CD64, and CD117 positive. Also APL is HLA-Dr negative which differentiates it from other AMLs which are HLA-Dr positive.

Early initiation of induction therapy ATRA before confirmation of diagnosis has changed the management of APL. APL is curable due to the initiation of ATRA. APL is considered a severe hematologic emergency due to its rapidly progressing bleeding diathesis and risk of intracerebral hemorrhage. Making a presumptive diagnosis based on the peripheral blood smear and bone marrow aspirate along with the patient history is important because the earlier that the patient begins therapy the better the outcome. ATRA and blood product support should be started as early as possible. APL blasts are highly sensitive to anthracyclines. Anthracycline chemotherapy with combination ATRA boasts remission rates of more than 90%. ATRA otherwise known as all-trans retinoic acid is a derivative of retinoic acid which reverses the differentiation block of APL blasts. Arsenic therapy with arsenic trioxide is approved in Europe and the United States for relapsed and refractory APL.

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Aggressive supportive therapy involves FFP, cryoprecipitate and platelets to maintain platelet levels greater than 30,000-50,000/uL and fibrinogen levels above 150 mg/dL. This regimen typically lasts during the first week of induction therapy while the coagulation disorder resolves.

There are significant adverse effects with therapy for APL. A common complication during induction therapy with ATRA or ATO (arsenic) is the development of hyperleukocytosis. APL differentiation syndrome is a life-threatening complication that develops a fever, edema/weight gain, respiratory distress, lung infiltrates, and pleural or pericardial effusions. Differentiation syndrome typically occurs within the first two weeks of the onset of therapy. Intravenous Dexamethasone is recommended immediately in the suspicion of APL differentiation syndrome. In mild cases of differentiation syndrome, ATRA or ATO therapy can just be interrupted and continued after symptoms regressed and when leukocyte counts decrease. Arsenic trioxide toxicity causes electrolyte shifts, particularly involving potassium and magnesium which to no surprise can alter ECG readings causing most commonly a QT interval prolongation. ATO therapy must be discontinued in severe prolongations due to the increased risk of cardiac arrhythmias. Documented chemotherapy adverse effects include the typical nausea and vomiting, increased infections, anemia, thrombocytopenia, increased bleeding tendencies which is exacerbated due to the coagulopathy associated with APL, and cardiac effects. With long-term chemotherapy there is an increased risk of drug-induced secondary malignancies.

Choice of treatment and timing of treatment is extremely important. As mentioned earlier it is very important to start induction therapy upon the first suspicion of APL, even before molecular confirmation occurs.

-Caleb

Case Study Mini-Series; Diagnosis

The patient was diagnosed with subclinical DIC because complications from Acute Promyelocytic Leukemia (APL)

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The characteristic chromosomal translocation of Acute Promyelocytic Leukemia is the break and fusion of the PML gene located on chromosome 15 and the RARA gene located on chromosome 17. This results in a t(15;17) which is detectable in more than 90% of cases. The PML gene has a physiological role in apoptotic pathways and in genomic stability. The t(15;17) breakpoint in PML can occur in three different sites; bcr1 within intron 6, bcr2 within exon 6, and bcr3 within intron 3 . The RARA receptor is active in different variations within each tissue and is important for granulopoiesis. The PML-RARA fusion transcripts impair signaling which is mediated by RARA and interact with proteins that leads to the delocalization of normal PML from its nuclear structures known as NBs. It is in this way that the PML-RARA oncoprotein negatively acts on the normal physiology of the native PML protein. APL is a subtype of AML that has distinctive morphological, biological and clinical characteristics. It is classified as AML-M3 in the French-American-British (FAB) classification system. The cure rate for APL is ~80-90% for patients who survive induction therapy with ATRA. Before ATRA, the 10-day survival rate with treatment was 9.4%. A high blast count was significantly associated with hemorrhagic events and fatality within the first 10 days. A high blast count and thrombocytopenia was associated with death within 24 hours upon admission and treatment. APL predominantly affects a wide spectrum of individuals between the ages 20 and 59 with no gender discrimination. 10-15% of all AML diagnosed in adults is APL, although it can be seen in distinct populations in a higher percentage. 28.2% of all AML diagnosed in Brazil is APL, and 20% of all AML in Venezuela is APL.

APL presents as a bleeding diathesis and coagulopathy. The more common hypergranular variant of APL presents with leukopenia while the less common microgranular variant tends to be more aggressive and presents with leukocytosis. The malignant promyelocytes have specific properties that interact with the host cells. Maligant APL cells express tumor associated procoagulants; Tissue factor (TF) and cancer procoagulant (CP). Tissue factor is an activator of coagulation and the relative expression is elevated significantly in patients with APL.

APL is characterized as a hyperfibrinolysis state. Fibrinolysis is normally activated by thrombin as the fibrin clot develops and coagulation comes to an end. Malignant promyelocytes highly express annexin-II. Annexin-II is a protein receptor that has a strong affinity to plasminogen and tissue-type plasminogen activator (tPA) which results in strong yield of plasmin which initiates fibrinolysis. Annexin-II is highly expressed in the cerebral microvascular endothelial cells explaining the high prevalence of intracerebral hemorrhage in patients with APL. Cytokine release of IL-1B and TNFa by malignant promyelocytes upregulate apoptosis and upregulate the expression of tissue factor on endothelial cells. It is also common for the cytokines to cause loss of the anti-coagulant cofactor thrombomodulin. These various factors lead to APL-associated coagulopathy commonly seen.

Patients with APL present low fibrinogen levels, low platelet count, and an elevated PT-INR, aPTT, and D-dimer. In DIC secondary to APL, fibrinogen survival is markedly decreased due to rapid consumption and the liver can’t produce the product fast enough. Sometimes more specialized tests are needed to diagnose the coagulopathy in APL. Levels of thrombin-antithrombin complex (TAT), prothrombin fragment 1 and 2, and fibrinopeptide A are all increased and all indicate coagulation activation. Decreased levels of plasminogen, and a-2-antiplasmin further support the hyperfibrinolysis state. Sometimes it is helpful to further evaluate the coagulation process and its components. Protein C and antithrombin III are synthesized in the liver and are relatively normal in APL associated coagulopathy unless the maligancy is accompanied by hepatic dysfunction.

The next installment of the mini-series will focus on the key points of what lead to the diagnosis, what I look for as a medical laboratory professional in aiding the doctor in the diagnosis, and how to treat appropriately.

 

-Caleb

 

37-year-old South American Male Case Study Mini-Series

The purpose of this mini-series is to get in the mind of a treating physician when a patient such as this presents to the clinic or the ED in this case. The first part of this series is the introduction of the case with case history and initial lab testing. Please don’t hesitate to leave comments on what you think the diagnosis is and what other confirmatory tests need to be done if any as well as what treatment should consist of. This is mean’t to stimulate a discussion and there are no wrong answers. I am in no way a physician or at that level or have that education. I am a student with a passion for molecular diagnostics and creating these cases is a good way for me to practice real life scenarios through careful and diligent research as well as help others who think the same way. This case is no way real and all lab values are made up to the best of my knowledge. If anything is incorrect please do not hesitate to email me or  leave a comment.

CASE:

A 37-year-old South American male presented to his annual physical with his primary care physician with general fatigue, decreased appetite and weight loss over the past three weeks. The patient mentioned to his physician that he has had multiple nosebleeds throughout the last few weeks, an occurrence of multiple a week. The patients past medical history is unremarkable. No family history of bleeding tendencies. He is not taking any prescription medication and denies use of recreational drugs and only social use of alcohol. His physician ordered a CBC and a prothrombin time/activated partial thromboplastin time (PT/aPTT). Results are in table 1.

Two days later the patient presented to the emergency room with fever and heavy fatigue, he explained to the attending that it has been hard to do anything the last few days, and has been bed-ridden. Physical exam revealed bilateral bruising on the upper arms and forearms with purpura and petechiae. The attending physician ordered a full coagulation panel, platelet function tests (Ristocetin cofactor assay), bleeding time test for vWD, and full CBC with peripheral blood smear analysis. Results are summarized in table 2.

Later that evening the patient developed a high fever, and back/flank pain and was moved to the ICU. Blood cultures, CRP and a procalcitonin was ordered, results are in table 3.

Positive cultures for Staphylococcus aureus were found after 48-96 hours and the patient was started on a course of vancomycin and monitored closely.

Patient results indicated he was pancytopenic with a hemoglobin of 9.7 g/dL (Ref. 13.5-18.0 g/dL) and RBC count of 3.7×10^3/uL (Ref. 4.20-6.00×10^6 uL) with severe thrombocytopenia at 37×10^3/uL (Ref. 150-450×10^3/uL).

Initial coagulation results revealed significantly elevated PT and aPTT. The bleeding time test along with the results from the RCO indicate platelet dysfunction or acquired inhibition of platelets by accelerated destruction. Platelet aggregation studies were normal. RCO studies indicate factor VIII inhibition or consumption.

The peripheral blood smear confirmed leukopenia and thrombocytopenia and revealed abnormal promyelocytes with abundant azurophilic granulation and multiple auer rods in bundles. RBC morphology showed schistocytes and fragmented cells.

The attending followed up by ordering a complete fibrinogen, D-dimer and a plasminogen panel. Results are in table 4.

The significantly elevated D-dimer, elevation in t-PA and u-PA in combination with the significant decrease in fibrinogen, and plasminogen levels indicates primary hyperfibrinolysis.

The attending sent a blood sample to the Blood Bank laboratory and asked for units of packed red cells, platelets, and fresh frozen plasma (FFP) to be transfused. With the additional blood components, the patient was able to regain control over the thrombocytopenia, hemoglobin, fibrinogen and coagulation factor levels.

A bone marrow aspirate was ordered including cytology, cytochemistry, immunophenotyping, FISH (Fluorescence in situ hybridization), cytogenetics (chromosomal analysis and FISH) and RT-PCR for PML/RARA quantification of transcripts. The attending started the patient on all-trans retinoic acid (ATRA) as induction therapy.

FISH revealed the PML-RARA fusion gene present which was later quantified and confirmed by RT-PCR. PCR sequencing revealed a bcr-3 PML-breakpoint. Chromosomal analysis of the bone marrow identified a t(15;17) classic translocation. Cytochemistry revealed intensely positive reacting cells to myeloperoxidase and Sudan black B. Immunophenotyping results are in table 5.

Table 1:

RBC: 4.10×10^6/uL             4.20-6.00×10^6/uL          

HGB: 12.9 g/dL                    13.5-18.0 g/dL        

HCT: 38.7%                          40-54%

MCV: 88 fL                            80-100 fL

MCH: 33.2 pg                        26-34 pg

MCHC: 32.3 g/dL                  32-36 g/dL

RDW: 13.5%                         11.5-14.5%

RETIC: 0.8%                          0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 6.3×10^3/uL              3.6-10.6×10^3/uL

NEUT: 3.6×10^3/uL             1.7-7.5×10^3/uL

LYMPH: 1.9×10^3/uL          1.0-3.2×10^3/uL

MONO: 0.7×10^3/uL           0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                  0.0-0.3×10^3/uL

BASO: 0                                 0.0-0.2×10^3/uL

PLT: 111×10^3/uL              150-450×10^3/uL

MPV: 7.3 fL                           7.0-12.0 fL

 

PT: 21 seconds                    11-14 seconds

aPTT: 37 seconds               25-35 seconds

Table 2:

RBC: 3.7×10^3/uL              4.20-6.00×10^3/uL

HGB: 9.7 g/dL                     13.5-18.0 g/dL                    

HCT: 28.9%                          40-54%

MCV: 71 fL                            80-100 fL

MCH: 31.8 pg                       26-34 pg

MCHC: 33.1 g/dL                 32-36 g/dL

RDW: 15.1%                        11.5-14.5%

RETIC: 2.3%                         0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 2.5×10^3/uL             3.6-10.6×10^3/uL

NEUT: 1.3×10^3/uL           1.7-7.5×10^3/uL    

LYMPH: 0.7×10^3/uL         1.0-3.2×10^3/uL

MONO: 0.3×10^3/uL          0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                 0.0-0.2×10^3/uL

BASO: 0.1×10^3/uL            0.0-0.3×10^3/uL

PLT: 37×10^3/uL               150-450×10^3/uL

MPV: 19.3 fL                       7.0-12.0 fL

 

Myeloblasts: 7%                  0%

Promyelocytes: 54%         0%

Myelocytes: 3%                    0%

Metamyelocytes: 5%           0%

Bands: 0%                             0%

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PT: 33 seconds                   11-14 seconds

aPTT: 63 seconds              25-35 seconds

BT: 13 minutes                   1-9 minutes

RCO: 30%                              50-150%

Platelet aggregation studies: Normal

Table 3:

Blood Cultures: POS Staph aureus          NEG

Procalcitonin: 0.25 ng/mL                        <0.15 ng/mL

CRP: 23 mg/L                                                0-10 mg/L

Table 4:

Fibrinogen: 67 mg/dL

Plasminogen: Reduced

a2-Antiplasmin: Reduced

t-PA: Elevated

u-PA: Elevated

D-Dimer: >19,000 ng/mL    

Table 5:

CD2                NEG

CD4                NEG

CD13              POS

CD14              NEG

CD16              NEG

CD19              NEG

CD33              POS

CD34              NEG

CD45              POS

CD56              NEG

CD64              POS

CD117            POS

HLA-DR         NEG

 

-Caleb

Transfusion Reactions

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The blood bank of any laboratory deals with a huge responsibility. They play a role in the initial compatibility testing of blood donor products and the recipient or patient serum. The patients serum contains naturally occurring antibodies or in certain circumstances where the patient has had a previous transfusion, the serum can contain alloantibodies that have been synthesized from previous donor blood products. Research has progressed suggesting that whole blood donor products are not as effective at replacing volume as individual components are. When a donor comes in and donates a pint of blood there are techniques that are used to separate the plasma from the blood products. Platelets are collected via an apheresis machine. When the plasma is separated out it must be frozen at >-20 degrees C within 8 hours of collection. When a patient needs plasma, it takes about 18-20 minutes to thaw and release. Fresh frozen plasma (FFP) has an expiration off 12 months.  Red blood cells are usually stored in a refrigerator at 1-6 degrees C. RBC products have an expiration of 42 days once collected. They can be frozen for 10 years if needed.

For transfusion purposes compatibility needs to be done correctly and cautiously. Platelets do not need to be ABO or Rh compatible, but if ample supply is available, its best to ABO match donors with the patient. Red blood cells absolutely need to be ABO and Rh compatible. If a compatible unit is not available then the hospital should use an O negative unit. O negative units are used as the universal donor. Plasma should be ABO compatible. Contrary to RBCs units, an AB plasma donor is considered the universal donor where in RBC products an O negative donor is the universal donor as mentioned previously. Plasma products contain the donors antibodies. When the donor is AB, they do not have anti-A, or anti-B. It is because of this principle that AB plasma is considered as the universal donor.

Even when every precaution is taken to ensure proper testing took place and compatibility testing was as objectively accurate as possible transfusion reactions can still take place. There is no way to 100% prevent them. Acute hemolytic reactions are typically the most severe and occur when ABO-incompatible blood is given. With acute hemolytic reactions fever and chills develop quickly, back and flank/pain (Renal failure) can occur with hemoglobinuria/hemoglobinemia. Bleeding and DIC can commonly be seen. Treatment is to stop transfusion immediately and volume replacement. Diuretics may be given, most commonly furosemide. Febrile non-hemolytic reactions are typically caused by transfusion of leukocytes that attack the recipient. A fever that is characterized as greater than 1 degree Celsius increase. The infusion of the leukocytes cause cytokine release such as IL-6, and TNF. Transfusion of HLA antibodies can occur as well. Antipyretics can be given to resolve. It is also recommended to infuse leukocyte reduced units in the future.

Bacterial contamination can occur which can cause sepsis. Typically there will be a rapid high fever with symptoms of rigor, shock and gastro symptoms. Bacterial contamination usually is able to be cultured from the donor bag along with the collection site. Antibiotics should be administered with support as necessary. A way to get around this is to leukoreduce donor units.

Transfusion-related Acute Lung Injury (TRALI) is an acute lung injury <6 hours after transfusion that presents with hypoxemia and lung infiltrates. The anti-HLA antibodies activate the PMNs in the lung endothelial which causes physiological stress. TRALI is 20% fatal, but treatment should be aggressive supportive care with fluids.

Acute afebrile reactions include allergic, anaphylactic, and transfusion associated circulatory overload (TACO) reactions. A typical urticarial or allergic reaction presents with localized hives/redness which is caused by a IgE hypersensitivity. Typical treatment includes antihistamines. Anaphylactic reactions are caused by anti-IgA antibodies in the recipient. Usually signifying that the recipient is also IgA deficient. Presents with hypotension, GI symptoms and fever with anti-IgA. Treatment is immediate epinephrine or transfusion with washed RBCs or platelets. TACO usually occurs with a history of cardiopulmonary disease with too rapid of blood infusion. High risk groups include the elderly and adolescents. TACO presents with dyspnea, and hypoxia during and after transfusion. Elevated BNP, JVD and BP. Treatment is to slow the rate of infusion and diuretics.

Delayed febrile reactions typically present greater than one week post transfusion. There is a positive DAT along with hyperbilirubinemia and evidence of a new alloantibody. Delayed febrile reactions are caused by a anamnestic response to re-exposure to red cell antigens. Treatment is support therapy. Graft-vs-host-disease (GVHD) is caused by cellular immune response by transfused T-lymphocytes versus the host or recipient. Presentation includes fever, diarrhea, skin rash. Treatment includes immunosuppressive therapy with supportive care. GVHD can be 90% fatal.

Delayed afebrile reactions include post transfusion purpura and iron overload. PTP is caused by a recipient antibody versus the absent platelet antigen (HPA-1a). There is a decrease in platelets, and increased bleeding. Treatment includes IVIG and plasma exchange. Its important to avoid platelet transfusion. Iron overload typically occurs when >100 units have been transfused. Liver, pancreas, and cardiac dysfunction occurs. Iron chelation is standard treatment.

All reactions are serious and should be treated as such. Its important to check for clerical error in pre-transfusion compatibility testing as that is the number one cause of transfusion related reactions.